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Originally published In Press as doi:10.1074/jbc.M400466200 on July 9, 2004

J. Biol. Chem., Vol. 279, Issue 39, 40882-40889, September 24, 2004
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Lipoteichoic Acid and Toll-like Receptor 2 Internalization and Targeting to the Golgi Are Lipid Raft-dependent*

Martha Triantafilou{ddagger}, Maria Manukyan§, Alan Mackie¶, Siegfried Morath||, Thomas Hartung||, Holger Heine§, and Kathy Triantafilou{ddagger}**

From the {ddagger}Infection and Immunity Group, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, United Kingdom, the §Department of Immunology and Cell Biology, Research Center Borstel, Parkallee 22, Borstel D-23845, Germany, the Department of Food Material Science, Institute of Food Research, Norwich Research Park, Norwich NR4 7UA, United Kingdom, and the ||Department of Biochemical Pharmacology, University of Konstanz, Konstanz D-78457, Germany

Received for publication, January 15, 2004 , and in revised form, July 6, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Lipoteichoic acid (LTA), a key cell wall component of Gram-positive bacteria, seems to function as an immune activator with characteristics very similar to lipopolysaccharide from Gram-negative bacteria. It has been shown that LTA binds CD14 and triggers activation via Toll-like receptor 2, but whether the activation occurs at the cell surface or internalization is required to trigger signaling has yet to be demonstrated. In this work we have investigated LTA binding and internalization and found that LTA and its receptor molecules accumulate in lipid rafts and are subsequently targeted rapidly to the Golgi apparatus. This internalization seems to be lipid raft-dependent because raft-disrupting drugs inhibited LTA/Toll-like receptor 2 colocalization in the Golgi. Similarly to lipopolysaccharide, LTA activation occurs at the cell surface, and the observed trafficking is independent of signaling.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Sepsis is an inflammatory response to infection either by Gram-negative or Gram-positive bacteria which can lead to multiorgan failure and death (1). This inflammatory response seems to be triggered by bacterial products, such as lipopolysaccharide (LPS)1 from Gram-negative bacteria, and lipoteichoic acid (LTA) from Gram-positive (2, 3). It is now well established that the triggering of the host response occurs at the level of innate defense, without the need for antigen presentation or the clonal expansion of cells to fight the invading pathogens. The recognition of these pathogen-associated molecular patterns is mediated through germ line-encoded receptors, called pattern recognition receptors.

At first glance it seems that both LPS and LTA activate the innate immune system via similar mechanisms of action. Both LPS and LTA bind CD14 (4–6), they activate signaling via Toll-like receptors (TLRs) (7–9), they activate various tyrosine kinases (10), and they both trigger NF-{kappa}B translocation (11). On second glance though it seems that there are differences in host responses that follow cell activation. Recent studies have reported significant differences in host responses to TLR2 versus TLR4 activation (12–16). Specifically, LPS has been reported to induce TNF-{alpha}, macrophage inflammatory protein-1{beta}, and RANTES production in human alveolar macrophages, whereas TLR2 agonists induced only macrophage inflammatory protein-1 production (17), thus suggesting that different ligands trigger distinct cellular responses even though they share the ability to activate NF-{kappa}B. The question remains as to how these differences in host response are achieved. It is possible that different ligands trigger distinct TLR-mediated signaling pathways. Although both TLR2 and TLR4 trigger the MyD88-dependent signaling pathway, via MyD88 (18, 19), IL-1 receptor-associated kinase (20), and tumor necrosis factor receptor-associated factor 6 (21) leading to NF-{kappa}B activation, TLR4 ligands are also able to trigger an MyD88-independent pathway. The MyD88-independent pathway seems to involve the toll/IL-1 receptor domain-containing adaptor protein (22) as well as TIR-domain-containing adaptor inducing IFNs (23) and leads to the activation of NF-{kappa}B as well as IFN regulatory factor 3 (24). The existence of individual signaling pathways for each TLR may explain the distinct biological responses elicited by the different ligands.

It is now clear that the mechanism of LPS activation seems to require the formation of a receptor cluster that comprises at least CD14, TLR4, and MD-2 (25). The possibility of the formation of even larger activation clusters has also been suggested (6, 26). Furthermore, it has been shown recently that this activation is triggered at the cell surface in myeloid cells, and LPS is subsequently dragged to the Golgi apparatus along with CD14 and TLR4 (27). In intestinal epithelial cells, LPS seems to trigger TLR4 activation intracellularly within the Golgi apparatus (28). In the case of LTA the mechanism is not as well understood. It has been shown that LTA binds CD14 (29) and triggers activation via TLR2 (7, 30), but whether the activation occurs at the cell surface or internalization is required to trigger signaling has yet to be demonstrated.

In this work we have chosen to investigate the cell surface interactions as well as the intracellular trafficking of bacterial LTA. Using green fluorescent protein (GFP)-TLR2 we investigated the localization of TLR2 and LTA at the cell surface, defined the subcellular localization and trafficking of LTA by employing fluorescent imaging methods. Here we show that LTA associates with TLR2 at the cell surface within lipid rafts. LTA is subsequently internalized and rapidly reaches the Golgi apparatus independently of the clathrin-interacting endocytic machinery. This transport pathway appears to be raft-specific, and receptors involved in LTA recognition, CD14 and TLR2, seem to follow it.

Overall, our data suggest that TLR2 and LTA follow the considerable fluidity of lipid rafts between the plasma membrane and the Golgi complex to gain access to the cell. Our findings are in agreement with Latz et al. (27) and Nichols et al. (31), who have demonstrated a novel rapid recycling pathway from the plasma membrane to the Golgi which is followed by LPS and lipid raft markers. It seems that LTA binding induces the recruitment of TLR2 into membrane microdomains and takes advantage of the internalization of these domains to reach and accumulate in the Golgi apparatus. Similarly to LPS, LTA trafficking is independent of signaling.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—LTA from Staphylococcus aureus was prepared as described previously (32). LysoTraker Red DND-99, TR-Dextran, TR-ConA, and BODIPY-TR-ceramide were obtained from Molecular Probes, Inc. EEA1-specific as well as clathrin-specific polyclonal sera were obtained from Santa Cruz. LTA-specific antibody was purchased from Cambridge Biosciences (Cambridge, UK). Hybridoma cells secreting 26ic (anti-CD14) and W6/32 secreting MHC class I-specific mAb were obtained from the American Type Culture Collection (ATCC). TLR2-specific antibody, TL2.1, was purchased from HyCult (Denmark). The antibodies were conjugated to either Cy3 or Cy5 using Cy3 and Cy5 labeling kits from Amersham Biosciences. Cholera toxin was purchased from List Laboratories. The following chemicals were obtained from Sigma and used to study the LTA entry procedures: 20 µM nocodazole for depolymerization of microtubules, 25 and 50 µM) chloropromazine to inhibit the formation of clathrin-coated pits, 25 µM nystatin and 1 µg/ml filipin for the disruption of rafts.

Expression Plasmids—The vector pcDNA3 (Invitrogen) was modified to include GFP as C-terminal epitope tag in-frame with a cloning site. PCR of TLR2 was performed on pRK7-TLR2 (obtained from Dr. C. Kirschning, Technical University of Munich) to construct chimeric fluorescent cDNA. The upper and lower primers for TLR2 were 5'-GAAGCAGGATCCATGCCACATACTTTGT-3' and 5'-GGGCTCGAGGGACTTTATCGCAGCTCTCAGA-3'. The PCR fragments were digested with BamHI and XhoI and cloned in-frame into pcDNA3-GFP. An expression plasmid containing huCD14 (pcDNA3-huCD14) was a kind gift from Dr. D. T. Golenbock (University of Massachusetts, Worcester).

Stable Cell Lines—Stable transfections of HEK293 cells with pcDNA3, GFP-TLR2, or CD14 were performed using Superfect Transfection Reagent (Qiagen) according to the manufacturer's recommendations. Positive selection by fluorescence-activated cell sorting was performed. Clonal cell lines were obtained by limiting dilution.

Stably transfected cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 0.5 unit/ml penicillin, 0.5 µg/ml streptomycin, 400 µg/ml G418 for HEK/pcDNA3, HEK/GFP-TLR2, HEK/CD14, HEK/GFP-TLR2/CD14, and 400 µg/ml hygromycin for HEK/GFP-TLR2/CD14.

The Chinese hamster ovary/CD14/huTLR2 (3E10TLR2) reporter cell line was constructed by stable cotransfection of 3E10 with the cDNA for human TLR2 and pcDNA3 (Invitrogen), as described previously (33). Chinese hamster ovary cell lines were grown in Ham's F-12 medium containing 10% fetal calf serum and 1% penicillin/streptomycin. The medium was supplemented with 400 µg/ml G418 and 400 µg/ml hygromycin (3E10TLR2).

Luciferase Reporter Assays for NF-{kappa}B Activation—HEK293 cells transfected with CD14 and GFP-TLR2 were seeded into 96-well plates. The following day, the cells were transiently transfected with a NF-{kappa}B luciferase reporter gene using LipofectAMINE 2000 (Invitrogen) according to the manufacturer's instructions. The next day the cells were stimulated as indicated, and after6hof stimulation the cells were lysed in passive lysis buffer (Promega). Luciferase activity was measured using a plate reader luminometer.

TNF-{alpha} ELISA—Stably transfected HEK/GFP-TLR2/CD14 cells were plated on 24-well dishes (Nunc, Germany) at a concentration of 1 x 106/ml in 300 µl of Dulbecco's modified Eagle's medium and 10% fetal calf serum. The next day cells were stimulated for the indicated time period. Supernatants were collected and analyzed for TNF-{alpha} with a commercial ELISA (Research Diagnostics, Inc.).

Cell Labeling for Fluorescence Resonance Energy Transfer (FRET)— Stably transfected HEK/GFP-TLR2/CD14 were seeded on microchamber culture slides (Lab-tek, Invitrogen) and labeled with 100 µl of a 1:1 mixture of donor-conjugated antibody (Cy3) and acceptor-conjugated antibody (Cy5). For control experiments to determine whether FRET is dependent on donor and acceptor surface density, the donor:acceptor ratio was varied to 1:2 and 1:4. The cells were rinsed twice in phosphate-buffered saline and 0.02% bovine serum albumin prior to fixation with 4% formaldehyde for 15 min. The cells were fixed to prevent potential reorganization of the proteins during the course of the experiment.

Fluorescent Imaging—HEK/GFP-TLR2/CD14 cells were grown on microchamber culture slides. Cells were stimulated with 1 and 10 µg/ml LTA, and after different times poststimulation they were exposed to fluorescent stains for organelles and subsequently fixed with 4% formaldehyde for 15 min. For LTA and receptor staining fluorescent probes were added and incubated for 60 min in phosphate-buffered saline, 0.02% bovine serum albumin, 0.02% saponin, then washed three times with phosphate-buffered saline. The slides were mounted on Prolong antifadant (Molecular Probes).

When specific inhibitory drugs were used, the chemicals (nocodazole, chloropromazine, nystatin, filipin) were added to the cell culture medium prior to stimulation for 30 min, and they were also present during the course of LTA stimulation.

Cells were imaged on a Carl Zeiss, Inc. LSM510 META confocal microscope (with an Axiovert 200 fluorescent microscope) using a 1.4 NA 63x Zeiss objective. The images were analyzed using LSM 2.5 image analysis software (Carl Zeiss, Inc.). Fluorescein isothiocyanate and TRITC were detected using the appropriate filter sets. Using typical exposure times for image acquisition (less than 5 s), no fluorescence was observed from a Cy3-labeled specimen using the Cy5 filters, nor was Cy5 fluorescence detected using the Cy3 filter sets.

In addition, cells were imaged on a CCD (charged cooled device) camera-based microscope system, consisting of a Carl Zeiss 200-inverted microscope with an Orca ER CCD camera (Improvision, UK). Openlab software was used to control the camera, whereas image analysis was carried out using Volocity software.

FRET Measurements—FRET is a noninvasive imaging technique used to determine molecular proximity. FRET can occur over 1–10-nm distances and effectively increases the resolution of light microscopy to the molecular level. It involves nonradiative transfer of energy from the excited state of a donor molecule to an appropriate acceptor. The rate of energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor. The efficiency of energy transfer (E) is defined with respect to r and R0, the characteristic Forster distance by Equation1.

(Eq. 1)
In the present study, FRET was measured using a method described previously (34, 35).

FRAP Measurements—FRAP measurements were performed as described previously (36, 37). Briefly, slides containing labeled cells were placed onto a temperature-controlled microscope stage (Physitemp model TS-4) and allowed to equilibrate to the desired temperature for FRAP measurements. After equilibration, the beam of an argon ion laser (Innova 100-10) was focused onto the desired area on the cell. The laser beam was of Gaussian cross-sectional intensity distribution, with a half-width at 1/e2 height of the laser beam at its point of focus equal to 1.24-µm spot radius. FRAP measurements were recorded and analyzed as described previously (36). For intracellular FRAP experiments, the laser beam was focused inside the cell rather than on the cell surface.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
GFP-TLR2 Fusion Proteins Are Able to Respond to LTA— HEK293 cells are normally unresponsive to LPS or LTA because they lack CD14, TLR4, and TLR2 expression. In this work we transfected HEK293 cells with CD14 or CD14 and GFP-TLR2 to be able to define the subcellular localization and trafficking of TLR2 before and after stimulation by bacterial LTA. HEK293 cells transfected with CD14 and GFP-TLR2 successfully expressed TLR2 on the cell surface (Fig. 1A), similarly to human monocytes (Fig. 1B).



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  		FIG. 1.
Fluorescent TLR2 is a functional receptor. HEK/GFP-TLR2/CD14 cells (A) and human monocytes (B) were labeled with anti-TLR2 (TL2.1) (white histograms) or isotype control IgG (gray histograms) followed by secondary APC-conjugated antibody and analyzed by flow cytometry counting 10,000 cells, not gated. HEK293, HEK/CD14, HEK/GFP-TLR2/CD14, and human monocytes were stimulated with either 10 µg/ml LTA (white bar charts) or 100 ng/ml LPS (gray bar charts) for 4 h. TNF-{alpha} was measured in the cell supernatant by ELISA (C).

 
To establish that transfection of HEK293 cells with CD14 and TLR2 rendered them able to respond to LTA, we measured TNF-{alpha} secretion in HEK293, HEK/CD14, and HEK/GFP-TLR2/CD14 cells following LTA stimulation.

HEK293, HEK/CD14, and HEK/GFP-TLR2/CD14 cells as well as human monocytes were stimulated with 10 µg/ml LTA. In parallel as a control HEK293, HEK/CD14, HEK/GFP-TLR2/CD14, and human monocytes were stimulated with 100 ng/ml LPS. After stimulation supernatants were collected and analyzed for TNF-{alpha} using a commercial ELISA.

As expected it was shown that HEK293 cells were incapable of responding to either LPS or LTA. Transfection with CD14 did not make them responsive to bacterial products, whereas transfection with CD14 and TLR2 rendered them responsive to LTA but not to LPS (Fig. 1C). Human monocytes expressing CD14, TLR4, and TLR2 (Fig. 1C) were able to respond to both LPS and LTA. From these data we conclude that transfection of HEK293 cells with both CD14 and TLR2 enables them to respond to LTA. In addition, the fluorescent tag fused on the C terminus of TLR2 does not seem to affect TLR2 function, and thus GFP-TLR2 is a functional signaling receptor for LTA.

Cell Surface Interactions of LTA: FRET Imaging of Lipid Rafts—It has been shown previously that the innate recognition of LPS takes place within lipid rafts or microdomains (38). Lipid rafts are believed to perform diverse functions by providing a specialized microenvironment in which the relevant molecules for the specific biological processes are partitioned from the rest of the plasma membrane and are concentrated into the raft (39). To investigate whether LTA recognition also occurs within lipid rafts FRET was utilized.

We measured FRET in terms of dequenching of donor fluorescence after complete photobleaching of the acceptor fluorophore. Increased donor fluorescence after complete destruction of the acceptor indicated that the donor fluorescence was quenched in the presence of the acceptor because of energy transfer. We tested the energy transfer efficiency in our system using as a positive control the energy transfer from mAbs Cy3-W6/32 and Cy5-MCA1115 to two different epitopes on MHC class I molecules (Table I), which showed that the maximum energy transfer efficiency (E) was 34 ± 2.5.


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  		TABLE I
Energy transfer efficiency values between donor-acceptor pairs on HEK cells Energy transfer between different pairs was detected from the increase in donor fluorescence after acceptor photobleaching. Data represent the mean ± S.D. of a number of independent experiments.

 
To visualize whether LTA was localized in lipid rafts we measured FRET on HEK/CD14/huTLR2 cells between LTA (using Cy3-LTA-specific mAb) and GM1 ganglioside, a raft-associated lipid (using Cy5-cholera toxin). Dequenching was observed once the Cy5 was photobleached (E = 24 ± 1.0%), suggesting that most (~62%) of the LTA concentrates in the lipid rafts of the plasma membrane (Fig. 2). Control experiments were performed with the Cy5-cholera toxin using Cy3-LTA-specific mAb without the presence of LTA. The results showed that the energy transfer efficiency was 1.2 ± 0.5%, thus verifying that there was no nonspecific interaction. In addition, control experiments were performed using the parental HEK293 cell line. It was found that barely detectable levels of LTA bound in the absence of CD14 (data not shown).



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  		FIG. 2.
TLR2 and GM1 ganglioside FRET measurements before and after LTA stimulation. Energy transfer between TLR2 (Cy3-TLR2) and GM1 ganglioside (Cy5-cholera toxin) before (A and B) and after stimulation by LTA (C and D) can be detected by the increase in donor fluorescence after acceptor photobleaching. A and C, donor (Cy3) image after acceptor photobleaching, B and D, E image (image resulting from the subtraction of the Cy3 image before photobleaching from the Cy3 image after photobleaching). E, E as a function of fluorescence, for D:A of 1:1 (triangles), 1:2 (open circles), and 1:4 (closed circles). Scale bar, 5 µm.

 
We similarly examined whether CD14 and TLR2 receptor molecules localize in lipid rafts prior to LTA stimulation, by using Cy3–26ic Fab specific for CD14 and Cy3-TLR2-specific mAb, respectively, and Cy5-cholera toxin for the GM1 ganglioside. Our results showed that there was FRET observed between CD14 and GM1 ganglioside (26 ± 1.5%), thus CD14 was present in lipid rafts prior to LTA stimulation (Table I). In contrast, there was no energy transfer between TLR2 and GM1 ganglioside prior to LTA stimulation (6 ± 1%), suggesting that similar to TLR4, TLR2 does not reside in lipid rafts prior to stimulation (Fig. 2B). Upon stimulation, we observed large dequenching (28 ± 2%) between TLR2 and GM1 ganglioside, thus suggesting that TLR2 is recruited within lipid rafts following LTA stimulation (Fig. 2D). As expected upon LTA stimulation, CD14 remained within the lipid raft because large dequenching was observed after Cy5-cholera toxin photobleaching. Control experiments investigating the association of TLR2 with GM1 ganglioside after LPS stimulation were also performed. It was shown that TLR2 was not recruited in lipid rafts following LPS stimulation because the energy transfer observed between TLR2 and GM1 ganglioside after LPS stimulation was 3 ± 0.5%. Similar results were obtained when human monocytes were utilized (Table II).


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  		TABLE II
Energy transfer efficiency values between donor-acceptor pairs on human monocytes Energy transfer between different pairs was detected from the increase in donor fluorescence after acceptor photobleaching. Data represent the mean ± S.D. of a number of independent experiments.

 
To rule out the possibility that the FRET observed was because of random distribution, we varied the ratio of donors and acceptors used to label the proteins of interest (Fig. 2E). E was found to be independent on acceptor density, to be sensitive to donor:acceptor ratio, and not to go to 0 at low surface density, thus suggesting that the FRET values observed were the result of clustered molecules and not random associations.

Entry Route—To investigate the entry route of LTA into cells, we used vital stains for known vesicular compartments in cells as well as fluorescent probes for LTA (using a Cy5-specific LTA mAb) and CD14 (Cy5–26ic Fab).

We determined whether LTA or its receptors CD14, TLR2 are internalized into lysosomes using a freely permeant probe with a high selectivity for acidic organelles, LysoTraker Red DND-99 (40). HEK/GFP-TLR2/CD14 were incubated for different time periods (5, 15, 30, and 60 min) with 1 and 10 µg/ml LTA in the presence of LysoTraker Red DND-99. Triple labeling with LysoTraker Red DND-99, LTA-specific probe, or CD14-specific probe and GFP-TLR2 was performed. Our results showed that LTA did not colocalize within lysosomes (data not shown).

Furthermore, we investigated the internalization of a fluid phase marker (TRITC-dextran), LTA, and GFP-TLR2. Fluorescent dextran, a hydrophilic polysaccharide with poly(a-D-1,6-glucose) linkages, labels early and late endosomes (41). LTA was found to concentrate in a compartment distinct from TRITC-dextran, showing us that LTA does not localize in late endosomes.

To determine whether LTA was localized in the Golgi we used two different markers to label compartments of the Golgi apparatus, Concanavalin A (ConA) and BODIPY-TR-ceramide. ConA is known to label the rough endoplasmic reticulum and the dilated cisternae of the cis-Golgi apparatus structure (42), whereas ceramide associates preferably with the trans-Golgi complex (43). Triple labeling with TRITC-labeled ConA or BODIPY-TR-ceramide, GFP-TLR2 and LTA at 5, 15, and 30 min following stimulation showed colocalization of the LTA and GFP-TLR2 with the Golgi after a 30-min stimulation (Fig. 3).



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  		FIG. 3.
TLR2 and LTA are localized in the Golgi. HEK/GFP-TLR2/CD14 cells were incubated with LTA and subsequently labeled with a Cy5-labeled anti-LTA antibody (blue) and TRITC-ConA vital stain for Golgi (red). The merged image shows extensive overlay of areas positive for Golgi, TLR2, and LTA (seen as white). Scale bar, 10 µm.

 
Effects of Clathrin Route Inhibitors on LTA Entry—To determine whether LTA and TLR2 utilize clathrin-coated pits for internalization we used different clathrin inhibitors prior to LTA stimulation. We utilized various chemicals that we had tested and were not cytotoxic to the cells, such as 20 µM nocodazole and 25 µM chloropromazine, as well as an anti-clathrin mAb. Nocodazole is known to block endosomal traffic between early and late endosomes (44), whereas chloropromazine causes the disappearance of clathrin-coated pits from the cell membrane (45).

Using triple fluorescent labeling we investigated the concentration of LTA as well as TLR2 in the Golgi apparatus after treatment with either nocodazole or chloropromazine. We found that neither nocodazole nor chloropromazine prevented the localization of LTA and TLR2 with the Golgi (Fig. 4B). Pretreatment of cells with these chemicals did not affect TNF-{alpha} secretion. Similar results were obtained when using an antibody specific for clathrin, suggesting that LTA/TLR2 entry into cells is clathrin-independent.



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  		FIG. 4.
Intracellular distribution of LTA and TLR2 after lipid raft disruption. HEK/GFP-TLR2/CD14 cells untreated (A) or pretreated with chloropromazine (B) or nystatin (C) were incubated with LTA and subsequently labeled with a Cy5-labeled anti-LTA antibody (blue) and TRITC-ConA vital stain for Golgi (red). The merged image shows extensive overlay of areas positive for Golgi, TLR2, and LTA (seen as white). Scale bar, 10 µm.

 
Effects of Raft-disrupting Drugs on LTA Entry—Because we had already found that LTA and its receptor molecules localize in lipid rafts in the initial binding steps we investigated whether lipid raft-disrupting drugs such as nystatin (46) or filipin (47), which had been tested and were not cytotoxic at the optimal concentrations of 25 µM and 1 µg/ml, respectively, could inhibit LTA/TLR2 targeting to the Golgi apparatus. Fluorescent labeling revealed that treatment with either filipin or nystatin prevented LTA and TLR2 colocalization to the Golgi (Fig. 4C), leading us to believe that LTA/TLR2 entry and targeting to the Golgi apparatus is dependent on lipid raft integrity. The cells viability was not affected either by nystatin or filipin treatment because the drug-treated cells excluded trypan blue.

To investigate whether the entire microdomain along with all the raft-associated receptors was being "dragged" to the Golgi apparatus we examined the translocation of Cy5-cholera toxin. HEK/GFP-TLR2/CD14 cells were incubated with Cy5-labeled cholera toxin, which binds to GM1 ganglioside (a lipid raft marker). It was shown that the cholera toxin was targeted rapidly from the cell surface to the Golgi apparatus indistinguishably from LTA, CD14, and TLR2 (Fig. 5).



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  		FIG. 5.
TLR2, CD14, LTA, and GM1 are targeted to the Golgi. HEK/GFP-TLR2/CD14 cells were incubated with LTA and subsequently labeled with a Cy5-labeled anti-LTA antibody (blue) and TRITC-ConA vital stain for Golgi (red) (A). HEK/GFP-TLR2/CD14 cells were labeled with a Cy5-labeled anti-CD14 antibody (blue) and TRITC-ConA vital stain for Golgi (red) (B) or with Cy5-labeled cholera toxin (blue) and TRITC-ConA vital stain for Golgi (red) (C). In the merged images colocalization of all three colors is seen as white. Scale bar, 10 µm.

 
TLR2 Intracellular Mobility—Because TLR2 was found to be targeted rapidly to the Golgi along with bacterial LTA, we employed FRAP to determine the intracellular TLR2 mobility before and after LTA uptake. FRAP would be able to provide us with a diffusion coefficient D as well as the extent of fluorescent recovery (mobile fraction). Thus we would be able to determine whether TLR2 associates with immobile or slow diffusing molecules intracellularly, if we observed a lower D after LTA stimulation. In addition we would be able to determine the percentage of molecules that are free to diffuse to and from the plasma membrane.

The intracellular diffusion of GFP-TLR2 prior to LTA stimulation was found to be (3.96 ± 1.0) x 10-8 cm2/s with a percentage recovery of 68 ± 10%, suggesting that similarly to TLR4, TLR2 is a highly mobile receptor that diffuses freely and rapidly through intracellular compartments. Upon LTA stimulation, the lateral diffusion of TLR2 was found to be (2.79 ± 1.0) x 10-8 cm2/s with a percentage recovery of 52 ± 8%, thus suggesting that upon LTA stimulation, TLR2 seems to be diffusing slightly slower possibly because of its association with other intracellular proteins. In addition we observed a reduction in percentage recovery, suggesting that a percentage of the TLR2 pool seems not to be able to diffuse freely, possibly because a small percentage (~10%) is being retained within the Golgi (Table III).


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  		TABLE III
Lateral diffusion of intracellular TLR2 The values represent the mean ± S.D. from several determinations (f10).

 
LTA Trafficking in the Presence and Absence of TLR2—To determine whether the trafficking and targeting of LTA and TLR2 to the Golgi apparatus were required for LTA signaling we proceeded to investigate the trafficking of LTA in the absence of TLR2.

In the absence of TLR2, in HEK/CD14 cells there was rapid LTA binding and internalization. LTA was targeted rapidly to the Golgi apparatus in the absence of TLR2. Using triple labeling we were able to visualize colocalization of CD14, LTA, and ConA (Fig. 6), thus suggesting that the intracellular targeting to the Golgi is not TLR2-dependent. In addition, CD14-mediated binding and uptake of LTA occurred in the absence of signaling because stimulation of HEK/CD14 cells with LTA did not result in MyD88 translocation (data not shown) or TNF-{alpha} secretion (Fig. 1).



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  		FIG. 6.
LTA internalization in the absence of TLR2. HEK/CD14 cells were incubated with LTA and subsequently labeled with a Cy5-labeled anti-LTA antibody (blue), and TRITC-ConA vital stain for Golgi (red). Intracellular CD14 was stained using 26ic antibody directly labeled with Oregon Green. Scale bar, 10 µm.

 
To examine further the significance of the LTA Golgi localization we disrupted the Golgi of human monocytes by adding brefeldin A (48). In brefeldin A-pretreated cells, Golgi was disrupted, and LTA disappeared from the perinuclear area (Fig. 7B). However, despite its relocation NF-{kappa}B activation in brefeldin A-treated cells was not diminished (Fig. 7C). Because LTA internalization and trafficking occurred in the absence of TLR2, and disruption of the LTA localization in the Golgi did not affect NF-{kappa}B activation, it is apparent that internalization and trafficking are neither sufficient nor necessary for LTA-induced activation.



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  		FIG. 7.
LTA-Golgi localization is not necessary for cellular activation. Human monocytes untreated (A) or treated with brefeldin A (B) were incubated with LTA and subsequently labeled with a Cy5-labeled anti-LTA antibody (blue) and TRITC-ConA vital stain for Golgi (red). Intracellular TLR2 was stained using TL2.1 antibody labeled directly with Oregon Green. Images were collected using a Zeiss 510 META confocal microscope. Scale bar, 10 µm. HEK/GFP-TLR2/CD14 cells expressing an NF-{kappa}B reporter construct were untreated or treated with 5 µg/ml brefeldin A for 2 h before stimulation with 10 µg/ml LTA or mock treated with medium for further 4 h. After stimulation, the cells were lysed and analyzed for luciferase activity (C). The data shown represent a mean of three independent experiments.

 
LTA Induces TLR2 Clustering—To verify that signal transduction is initiated from the plasma membrane, we also investigated the cell surface TLR2 distribution in response to LTA. HEK/GFP-TLR2/CD14 cells were stimulated with 10 µg of LTA and imaged by confocal microscopy. It has been shown that upon LTA stimulation, cell surface TLR2 seemed to form large clusters (Fig. 8). To investigate whether signaling was initiated via these clusters, we analyzed the location of MyD88 before and after LTA stimulation. Prior to LTA stimulation, MyD88 was found to be expressed in cytoplasmic aggregates (Fig. 8A). Upon LTA stimulation, it was found that MyD88 was recruited toward the cell surface and colocalized with the TLR2 clusters (Fig. 8B), thus suggesting that signaling is initiated via clustering of receptor molecules on the cell surface.



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  		FIG. 8.
LTA induces TLR2 clustering. HEK/GFP-TLR2/CD14 cells were either not stimulated (A) or stimulated with 10 µg of LTA (B) and imaged using a Zeiss 510 META confocal microscope. Intracellular MyD88 was stained using a polyclonal antibody directly labeled with TRITC. Merged images showing extensive overlay of areas positive for TLR2 and MyD88 are seen as yellow. Scale bar, 10 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The increased prevalence of Gram-positive sepsis has sparked an interest in identifying the molecular mechanisms that are involved in the host response to components of these organisms. LTA, a key cell wall component of Gram-positive bacteria, seems to function as an immune activator with characteristics very similar to LPS. Although these two bacterial components seem to induce the same end result (49), there seem to be significant difference between sepsis caused by Gram-positive and Gram-negative bacteria, mainly in the host responses against them (50). In this work we have chosen to investigate whether LTA binding and internalization are similar to those that have recently been reported for bacterial LPS (27).

To perform this work we engineered fusion protein of GFP with TLR2. We transfected HEK293 cells with CD14 and GFP-TLR2 and demonstrated that GFP-TLR2 is a fully functional receptor for bacterial LTA and triggers identical immune responses with cells that natively express TLR2.

Initially we looked at the distribution on the plasma membrane of LTA, CD14, as well as TLR2, to determine whether they reside in membrane microdomains or lipid rafts. Using FRET we found that LTA as well as both CD14 and TLR2 are concentrated within lipid rafts on the plasma membrane upon LTA stimulation. CD14, which is a glycosylphosphatidylinositol-linked protein, seems to be constitutively localized in lipid rafts (38, 51), whereas TLR2 was found not to reside in lipid rafts prior to LTA stimulation, but to accumulate increasingly in the raft upon the presence of LTA.

After LTA binding we attempted to track the entry route of LTA and its receptors within the cell. To elucidate LTA entry, we utilized triple labeling fluorescent imaging to visualize LTA, TLR2, and markers for different intracellular compartments. We found that LTA and TLR2 did not colocalize with lysosomes, endosomes, or endoplasmic reticulum over different time periods after LTA stimulation. In contrast it was found that both LTA and TLR2 were targeted rapidly to the Golgi apparatus and specifically to the cis-Golgi network 30 min after LTA stimulation. CD14 followed the same route. These data are in good agreement with previous studies performed with bacterial LPS (27, 52).

In an attempt to verify whether the entry of LTA and its receptor molecules was dependent on clathrin-coated pits we utilized different clathrin inhibitors prior to stimulation with LTA. Anti-clathrin-specific antibody as well as chemicals such as nocodazole and chloropromazine, which are known to block endosomal traffic between early and late endosomes (44) and to cause the disappearance of clathrin-coated pits from the cell membrane (45), respectively, were utilized. Neither the chemicals nor the mAb could inhibit LTA/TLR2 internalization. Furthermore, using triple labeling we visualized LTA, TLR2, and Golgi markers. We found that preincubation with the clathrin-route inhibitors did not affect LTA/TLR2 targeting to the Golgi, thus suggesting that LTA along with TLR2 gain entrance and are targeted to the Golgi via a clathrin-independent route.

In addition, LTA internalization seemed to be lipid raft-dependent. In the presence of lipid raft-disrupting drugs, such as nystatin or filipin, targeting of LTA and TLR2 to the Golgi network was inhibited upon disruption of the lipid raft integrity. Our studies suggest that lipid raft formation is crucial for LTA internalization and trafficking. Previous observations have shown that the entire cholesterol, ganglioside-rich microdomain or "lipid raft," where we have shown that LTA, CD14, and TLR2 reside, is constantly shuttling back and forth from the cell surface to the Golgi (31, 53, 54). Our studies confirm this because when we incubated HEK/GFP-TLR2/CD14 cells with Cy5-labeled cholera toxin, which binds to GM1 ganglioside (a lipid raft marker), the cholera toxin was targeted rapidly from the cell surface to the Golgi apparatus indistinguishably from LTA, CD14, and TLR2.

Our data suggest that LTA is dragged to the Golgi along with the lipid raft containing CD14 and TLR2 via a novel clathrin-independent endocytic route. There seems to be a plasma membrane-Golgi cycling pathway that is followed by sphingolipids that make up lipid rafts.

When we utilized FRAP to investigate TLR2 intracellular mobility before and after LTA stimulation, we found that prior to LTA stimulation TLR2 exhibited a very fast diffusion coefficient and a high percentage recovery indicative of a molecule that diffuses rapidly without any barriers throughout intracellular compartments. Upon LTA stimulation, TLR2 exhibited a slower diffusion coefficient and a lesser percentage recovery, suggesting that upon stimulation by LTA, TLR2 seemed to associate with other intracellular proteins and/or to be retained in the Golgi (the compartment that it seems to be residing in).

The question that we had to answer was whether the internalization and trafficking that we observed were crucial for LTA signaling. Over the years it had been suggested that this might be the case for LPS-induced signaling (28, 52), although it has recently been shown that LPS internalization is independent of signal transduction (27). To answer this question in the case of LTA, we performed confocal experiments with HEK/CD14 cells that were shown not to be able to respond to bacterial LTA. It was found that LTA was internalized and targeted rapidly to the Golgi in the absence of TLR2. Brefeldin A disruption of the Golgi showed that LTA/TLR2 localization in the Golgi is not essential for signaling and cytokine secretion. On the contrary we found that similarly to LPS (27), LTA must engage its receptors on the cell membrane where signaling is initiated. Thus our data are in agreement with Latz et al. (27) who have demonstrated that LPS internalization is independent of signal transduction, but we are in contrast to a recent report by Blander and Medzhitov (54) who showed that the internalization of Escherichia coli to the phagosome is dependent on TLR signals. The reason for these conflicting reports might be the fact that Blander and Medzhitov used whole bacteria and not bacterial products, such as LPS or LTA, in their study. It is possible that whole bacteria, because of the difference in particle size, are internalized by a different mechanism when compared with bacterial products.

Our work, which utilizes bacterial products, suggests that TLR2 forms clusters in response to LTA, most likely within lipid rafts, which result in signaling. It is possible that within these clusters other molecules might associate with TLR2 because it has been shown previously that it can heterodimerize with either TLR1 or TLR6 in response to different pathogens (55–57). Once TLR2 has been engaged by LTA, clustering occurs, which leads to signaling. Subsequently the entire microdomain, along with the receptors that have clustered there, is internalized and targeted to the Golgi. One possible explanation for this could be that after cellular activation, the plasma membrane is reorganized, TLR molecules are internalized to be recycled back to the surface, and LTA is targeted to the Golgi for clearance. Thus the process of internalization and intracellular trafficking of LTA is completely independent of signaling and is consistent with the hypothesis that cellular activation in response to bacterial products occurs at the cell surface.


   FOOTNOTES
 
* This work was supported by the Wellcome Trust (to K. T.) and German Research Foundation Grant He 2758/3-1 (to H. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

** To whom correspondence should be addressed: School of Life Sciences, University of Sussex, JMS Bldg., Falmer, Brighton BN1 9QG, United Kingdom. Tel.: 44-1273-678-362 or 923; Fax: 44-1273-678-362; E-mail: K.Triantafilou{at}sussex.ac.uk.

1 The abbreviations used are: LPS, lipopolysaccharide; ConA, concanavalin A; ELISA, enzyme-linked immunosorbent assay; FRET, fluorescence resonance energy transfer; GFP, green fluorescent protein; GM1, Il3NeuAc-GgOse4Cer; HEK, human embryonic kidney; LTA, lipoteichoic acid; mAb, monoclonal antibody; MHC, major histocompatibility complex; RANTES, regulated on activation normal T cell expressed and secreted; TLR, Toll-like receptor; TNF-{alpha}, tumor necrosis factor-{alpha}; TRITC, tetramethylrhodamine isothiocyanate; FRAP, fluorescence recovery after photobleaching. Back


   ACKNOWLEDGMENTS
 
We thank Professor Jon Cohen for a critical reading of this manuscript.



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M. Triantafilou, F. G. J. Gamper, R. M. Haston, M. A. Mouratis, S. Morath, T. Hartung, and K. Triantafilou
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F.-S. X. Yu and L. D. Hazlett
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