Published online before print May 5, 2008
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* Norwegian University of Science and Technology, Institute of Cancer Research and Molecular Medicine, Trondheim, Norway;
University of Konstanz, Biochemical Pharmacology, Konstanz, Germany;
University of Oslo, Department of Molecular Biosciences, Oslo, Norway;
European Commission of Joint Research Centre, Institute for Health and Consumer Protection, European Centre for the Validation of Alternative Methods, Ispra, Italy; and
¶ University of Massachusetts Medical School, Department of Medicine, Division of Infectious Diseases and Immunology, Worcester, Massachusetts, USA
1Correspondence: Institute of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Olav Kyrres gt. 9, N-7489 Trondheim, Norway. E-mail: terje.espevik{at}ntnu.no
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B activation. LTA internalization, but not NF-B 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.
Key Words: TLR • LTA • endocytosis
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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TLRs recognize a range of pathogen-associated molecular patterns (PAMPs), such as LPS and LTA. TLRs further initiate proinflammatory responses required for clearance of infection by the same mechanisms that potentially cause sepsis [7
]. Thirteen mammalian TLRs (TLR1–13), which recognize different PAMPs, have been identified to date [7
]. These are germ-line-encoded transmembrane proteins, consisting of an extracellular leucine-rich repeat domain and a cytoplasmic domain sharing homology with the mammalian IL-1R [8
, 9
]. A signaling cascade initiated by activation of the TLRs results in translocation of the transcription factor NF-B, which subsequently induces the expression of TNF, IL-1β, IL-6, and IL-8 and maturation of APCs [10
]. Although TLR4, in complex with the small, secreted glycoprotein myeloid differentiation protein 2 (MD2), recognizes LPS from Gram-negative bacteria [11
, 12
], TLR2 recognizes a particularly broad range of ligands, including Gram-positive bacteria and cell-wall components such as LTA as well as peptidoglycan and lipoproteins [13
14
15
16
17
18
]. Additional TLR2 ligands may include zymosan, glycolipids from spirochetes,lipoarabinomannan, and porins from Neisseria, among others [19
]. The ability of TLR2 to recognize such a wide repertoire of ligands is partially explained by heterodimerization of TLR2 with TLR1 and TLR6. TLR2/TLR1 heterodimerization occurs in response to triacylated lipopeptides, such as N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2R,S)-propyl]-(R)-cysteinyl-seryl-(lysyl)3-lysine (Pam3CysSK4) [20
, 21
], and an optimal response toward diacylated lipopeptides is attained by heterodimerization of TLR2 with TLR6 [22
, 23
]. LTA is recognized by the TLR2/TLR6 heterodimer, supposedly as a result of the two diacyl chains in the molecule [24
].
The monocyte differentiation antigen CD14 is a GPI-linked receptor expressed by cells of the monocytic lineage [25 , 26 ]. The receptor is shown to be highly concentrated in lipid raft microdomains of these cells [27 ]. Soluble CD14 and LPS-binding protein in serum transfer LPS to membrane-bound CD14, which further presents LPS to the TLR4/MD2 signaling complex [28 29 30 31 ]. The entire complex has further been shown to shuttle between the plasma membrane and the Golgi, independent of signaling, which is believed to occur predominantly at the plasma membrane [32 , 33 ]. CD14 has further been shown to bind lipopeptides and LTA in a similar manner [34 35 36 ]. The multifunctional B class scavanger receptor CD36 has, however, also been found to be involved in immune responses to TLR2/TLR6 ligands such as LTA in a manner analogous to CD14 [37 ]. Whether LTA binding to the plasma membrane is sufficient to induce signaling through TLR2 or whether internalization of the ligand is required is still under debate. Although some reports support that signaling occurs in lipid rafts, independent of ligand internalization [36 , 38 ], other reports show that reduced internalization of S. aureus and its component LTA correlates with diminished inflammatory response [39 ]. The relative role of CD14 and CD36 in response to LTA is furthermore in question with regard to whether the coreceptors participate in the same TLR2/TLR6 signaling complex or whether they enhance TLR2-mediated responses independent of one another.
In this study, we explored the uptake and trafficking pattern of LTA from S. aureus in relation to subcellular expression of TLR2 and its coreceptors CD36 and CD14 in human monocytes. We found TLR2 expressed in the plasma membrane, endosomes, lysosomes, and in Rab-11-positive compartments but not in the Golgi apparatus or the endoplasmic reticulum (ER). LTA rapidly accumulated in early and late endosomes, lysosomes, as well as in the ER and Golgi. CD14 and CD36 were required for optimal LTA binding/internalization and TNF release in monocytes. We further found that LTA internalization, but not NF-B activation, was inhibited in Dynamin-I K44A dominant-negative transfectants, showing that LTA is internalized by receptor-mediated endocytosis but that internalization is not required for signaling. These results support the hypothesis that signaling in response to LTA preferentially occurs at the plasma membrane, is independent of internalization, and requires CD36 and CD14 as coreceptors for TLR2.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1 molecule rhodamine or fluorescein per LTA. The labeled LTA was negative in the Limulus test for Gram-negative endotoxin (<0.1 pg/µg). LPS was from Escherichia coli strain O111:B4 and purchased from Invivogen (San Diego, CA, USA). Synthetic Pam3CysSK4 was purchased from EMC Microcollections (Tübingen, Germany). Antibodies used were anti-TLR2 (TL2.1) [18
], anti-TLR4 (HTA125), purified from hybridoma cells, kindly provided by Dr. Kensuke Miyake (Saga Medical School, Japan) [40
], unconjugated and FITC-conjugated anti-CD36 (FA6-152; Immunotech, France), anti-CD14 mAb 3C10 [41
] and 5C5 [42
], and MEM-18 (HyCult Biotechnology, Uden, The Netherlands). Additional antibodies used include mouse IgG and a Tricolor (PE-Cy5)-conjugated goat anti-mouse secondary antibody (Invitrogen, Carlsbad, CA, USA), anti-lysosome-associated membrane protein-1 (LAMP-1; R&D Systems, Minneapolis, MN, USA), anti-Golgin-97 (CDF4; Invitrogen), anti-Rab-11A (Abcam, UK), anti-GM130, anti-early endosomal antigen-1 (Eea-1), and unconjugated and FITC-conjugated anti-calnexin and mouse IgG (BD Biosciences, Franklin Lakes, NJ, USA). Alexa-conjugated antibodies were generated by protein labeling with Alexa 488 (A488), A546, or A647, according to the manufacturer’s instructions (Invitrogen). A633-labeled transferrin was purchased from Invitrogen. The following expression vectors were used: pcDNA3 (Invitrogen), human CD14 and TLR2-yellow fluorescent protein in pcDNA3 [43
], and Eea-1 tagged with GFP (Eea-1GFP) [44
]. The expression vector pORF9 and human CD36 in pORF9 were purchased from Invivogen. MD-2 in the expression vector pEF-BOS was kindly provided by Dr. K. Miyake [45
]. ERGFP encoding the ER-targeting sequence of calreticulin fused to cyan fluorescent protein (CFP; Clontech, Palo Alto, CA, USA). Dynamin-I wild-type and Dynamin-I K44A in pcDNA3 were kindly provided by Dr. Sandy Schmid (The Scripps Research Institute, La Jolla, CA, USA). Transient transfections were performed using GeneJuiceTM transfection reagent (Novagen, Darmstadt, Germany), according to the manufacturer’s instructions if not stated otherwise.
Cells and cell lines
Human monocytes were isolated from PBMC by adherence. PBMC were seperated from A+ buffy coats (Blood Bank, St. Olav’s Hospital Trondheim, Norway) using Lymphoprep, as described by the manufacturer (Axis-Shield, Norway). Monocytes were allowed to adhere in RPMI supplemented with 5% or 10% pooled A+ serum (St. Olav’s Hospital Trondheim) for 1 h at 37°C, 5% CO2, before cells were washed three times and fresh medium added. Human epithelial kidney 293 (HEK293) cell lines expressing TLR2 or TLR2 in combination with CD14 [43
] were cultured in DMEM supplemented with 10% FBS and the selection antibiotic G418 (0.5 mg/ml). Untransfected HEK293 cells were cultured in 10% FBS/DMEM. For confocal imaging, cells were seeded on 35 mm glass-bottom 
-irradiated tissue cell dishes (MatTek Corp., Ashland, MA, USA). Madine Darby canine kidney (MDCK) cells stably expressing Eea-1GFP were grown in DMEM supplemented with 9% FBS, 2 mM glutamine, 25 U/ml penicillin, and 25 µg/ml streptomycin at 37°C, 6% CO2.
Live microscopy
MDCK cells stably expressing Eea-1GFP were transiently transfected with the TLR2 overnight in Microwell dishes for microscopy (MatTek Corp.) using Lipofectamin, according to the manufacturer’s protocol (Invitrogen). Preceding imaging the cells, microscopy medium, DMEM without phenol red, and sodium carbonate supplemented with 3.5 g/L D-glucose to a final concentration of 4.5 g/L and 25 mM HEPES with 10% FBS were added. Cells were kept on ice for 45 min and then incubated with A546-conjugated anti-TLR2 mAb TL2.1 on ice for an additional 45 min. Image acquisition was performed on an Olympus Fluoview 1000 at 37°C with an Olympus PlanApo 60x/1.42 oil objective. Internalization analysis was carried out with ImageJ software, measuring the intensity of conjugated TLR2 as a function of time.
Confocal microscopy of subcellular expression of TLR2 and LTA internalization
Freshly isolated, live monocytes were added fresh RPMI medium supplemented with 0.1% A+ serum, incubated with LTArhodamine (20 µg/ml), and internalization of the ligand was followed by confocal microscopy at 37°C for 1 h. Freshly isolated monocytes were left unstimulated or stimulated with LTArhodamine for 1 h, 37°C, 8% CO2, prior to fixating with 4% paraformaldehyde, 10 min on ice, and then with 0.5 M K-PIPES/0.5 M EGTA/1 M MgCl2buffer, 10 min, at room temperature. Cells were then permeabilized with 50 mM NH4Cl/0.05% BSA/0.05% saponin, 20 min, at room temperature. Cells were subsequently stained in 50 mM NH4Cl/0.05% BSA/0.05% saponin, 20 min, at room temperature. Cells were also stained with A488- or A647-conjugated anti-TLR2 and antibodies against early endosome marker Eea-1FITC, lysosome marker anti-LAMP-1A647, trans-Golgi marker anti-Golgin-97A647, and cis-Golgi marker anti-GM130, anti-CD36FITC, anti-CD14A647 (3C10), or anti-CD14A488 (5C5). Freshly isolated monocytes were additionally stained intracellularly, as described, with anti-Rab-11A and subsequently with secondary antibody goat anti-rabbitA647, prior to staining with TL2.1A488. HEK293 cells were stained intracellularly after fixing cells with 4% paraformaldehyde for 10 min on ice and permeabilization with 20% A+/0.1% saponin/PBS for 20 min at room temperature. Cells were stained in 2% A+/0.1% saponin/PBS for 45 min at room temperature using 2–10 µg/ml antibodies before cells were washed and PBS added. Cells were observed by confocal microscopy using an Axiovert 100-M inverted microscope (Zeiss, Thornwood, NY, USA), equipped with an LSM 510 laser-scanning unit and a 63x 1.4-NA plan Apochromat oil-immersion objective (Zeiss). Appropriate filters were selected for the individual stainings.
A quantitative measure of colocalization of flourochromes was determined using the colocalization module of Imaris 5.0.2, 64-bit version (Bitplane AG, Zürick). "Percentage of material colocalized above threshold" of each subcellular marker or CD14 or CD36 that colocalized with TLR2 or LTA was calculated. This value takes into account the number of pixels that colocalizes, as well as the intensities ("material") of the two labels in each pixel. User-defined thresholds were set conservatively in a rectangular selection mode chosen above the apparent noise level for each channel. Colocalization maps showing colocalization events of each marker or CD36 or CD14 with TLR2 or LTA were created in Imaris. In these images, white denotes colocalization events between two channels, and pixels above the threshold that failed to colocalize were set to zero (black).
LTA-binding/internalization studies
A+ buffy coat from healthy donors (St. Olav’s Hospital Trondheim) was incubated with LTARhodamine Green (10 µg/ml) for 45 min at 4°C or at 37°C, 8% CO2. Incubation at 4°C should permit LTA binding to receptors on the plasma membrane but delay LTA internalization, and binding and internalization of LTA in monocytes were expected to proceed normally at 37°C. Erythrocytes were lysed with formic acid-based lysis buffer for 1 min, neutralized, and fixed using the Coulter Immunoprep Epics leukocyte preparation system (Coulter, Miami, FL, USA). Samples of cells were stained with fluoroscein (FITC)- or PE-conjugated mAb against CD14, CD3, or CD19. Samples were analyzed by flow cytometry, and populations were gated by CD14 high expression (monocytes), CD14 low expression (granulocytes), and CD3 or CD19 expression (lymphocytes), as well as by size and granularity. Gates were subsequently applied to determine LTARhodamine Green internalization in the samples by determining median fluorescence of each population. Monocytes were also stimulated by plating cells in sterile 96-plate wells coated overnight at 4°C with titrations of LTA or PBS or stimulated by adding titrations of LTA in solution or medium. Wells were washed four times with PBS after coating and prior to addition of cells to remove excess, unbound LTA. Cells were stimulated overnight in 1% A+/RPMI at 37°C, 5% CO2, before supernatant was harvested, and TNF levels were assessed by ELISA.
Cell staining for flow cytometry
Freshly islolated monocytes were detached with 0.02% EDTA, fixed with 4% paraformaldehyde, and stained extracellularly in 1% FBS/PBS with anti-TLR2A488, CD36FITC, CD14A488 (5C5), or mouse IgGA488/FITC and analyzed by flow cytometry. Freshly isolated monocytes were furthermore left unstimulated or stimulated for 16 h with LTA (0.1, 1, 10, 100, or 1000 ng/ml) prior to staining with anti-TLR2 or mouse IgG and subsequently, with secondary antibody goat anti-mouseTricolor, CD36FITC, or mouse IgG FITC, as described.
Luciferase reporter assay
NF-B activation was determined by a NF-B luciferase reporter assay as described previously [43
]. Briefly, HEK293 cells were transiently transfected with reporter plasmid endothelial leukocyte adhesion molecule-luciferase reporter gene (ELAM-Luc), containing a NF-B-dependent portion of the ELAM promoter-driving luciferase. Cells were additionally transfected with control plasmids pcDNA3 and pORF9 and/or CD36, CD14, and TLR2; CD36, CD14, and TLR2 in combination; or TLR4, CD14, and MD2 for 24 h. The total amount of each vector was kept constant by filling up with the appropriate control plasmids. Cells were subsequently stimulated with LTA (5 µg/ml) or LPS (100 ng/ml) for 5 h before cells were lysed and assayed for luciferase activity as a measure for NF-B activation using the luciferase assay system (Promega, Madison, WI, USA). Similar results were obtained upon repeating the experiment and including the pRL-TK Renilla luciferase control plasmid (Promega) as a control for transfection efficiency and cell viability in the setup. The dual luciferase assay system (Promega) was used to measure ELAM-Luc and Renilla luciferase activity, which was found to be similar in all wells (not shown).
Blocking studies
Freshly isolated monocytes were preincubated with mouse IgG, anti-TLR2, anti-CD36, anti-CD14 (MEM-18), or anti-TLR4 (10 ug/ml) in RPMI on ice (0–4°C) for 45 min and subsequently added LTARhodamine Green (2 µg/ml) for 45 min on ice. Cells were detached with 0.02% EDTA/PBS, washed, and analyzed for LTA binding and internalization by flow cytometry. Monocytes were pretreated with optimized concentrations of anti-CD36 (0.5 µg/ml), anti-CD14 (3C10; 10 µg/ml), a mixture of TL2.1 and TL2.3 (10 µg/ml), or mouse IgG (10 µg/ml) for 45 min at room temperature prior to stimulation with medium, LTA (10 µg/ml), LPS (20 ng/ml), or Pam3CysSK4 (50 ng/ml) in 1% A+/RPMI for 5 h at 37°C, 5% CO2, before the supernatant was harvested and analyzed for TNF by ELISA (R&D Systems).
Dynamin-I expression studies
HEK293-TLR2 cells were transiently transfected with wild-type Dynamin-I or the mutant Dynamin-I K44A in the presence or absence of transfected CD14 for 72 h before cells were incubated with LTArhodamine (2 µg/ml) and transferrinA633 (2 µg/ml) for 30 min at 37°C and observed by confocal microscopy. HEK-TLR2 cells were transiently transfected as described with the ELAM-Luc, combinations of CD14, Dynamin-I wild-type or the mutant Dynamin-I K44A, and/or control pcDNA3 for 72 h. Cells were subsequently stimulated with medium or LTA (5 µg/ml) for 5 h before cells were lysed and analyzed for NF-B activation.
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Figure 1. Surface TLR2 is internalized into endosomes. (A) Confocal images of internalization of TLR2 mAb (red) in MDCK cells expressing Eea-1GFP (green) and TLR2. Images show enlargements of a portion of a representative cell at 4-s interval time-points (two upper panels). Arrows denote an EEA-1GFP (green) endosome containing TLR2 mAb (red) that matures and loses the early endosome tag. The full picture of the cell (lower picture) shows TLR2 mAb internalization after 612 s. Cells were transiently transfected with TLR2 24 h prior to the experiment using Oligofectamine transfection reagent. Cells were kept on ice for 45 min and then incubated with TLR2A546 mAb on ice for 45 min. Image-acquiring was initiated 15 min post-incubation. Image acquisition was performed on an Olympus Fluoview 1000 at 37°C with an Olympus PlanApo 60x/1.42 oil objective. (B) Plot of relative intensity of total TLR2A546 fluorescence (black) and colocalization of TLR2A546 mAb with Eea-1GFP (green) and LysotrackerGreen (red) as a function of time. Results show measurements from five representative cells. Internalization analysis was carried out with ImageJ software. Dotted lines denote polynomial trend lines.
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TLR2 is expressed in the plasma membrane, endosomes, lysosomes, and Rab-11-positive compartments in monocytes
We further applied confocal microscopy to investigate the intracellular expression of TLR2 in freshly isolated monocytes to assess the subcellular compartments where LTA-induced signaling could occur. In line with previous studies [46
], TLR2 was found to be highly expressed in the plasma membrane of monocytes (Fig. 2
), as well as in a population of early endosomes (Fig. 2A)
and lysosomes (Fig. 2B)
. Furthermore, we found that TLR2 was not expressed in the Golgi network, using antibodies against Golgin-97 (Fig. 2C)
and GM130 (not shown), which are specific markers for the trans- and cis-Golgi, respectively [47
, 48
]. TLR2 expression was, however, often observed in close proximity to the trans-Golgi network, which led us to examine whether TLR2 was expressed in Rab-11A-positive compartments. The GTPase Rab-11A localizes to pericentriolar recycling endosomes and trans-Golgi and is essential for development of multivesicular body endosomal compartments [49
50
51
52
]. TLR2 colocalized with Rab-11A in the perinuclear area (Fig. 2D)
. These results suggest that TLR2 may also be expressed in recycling endosomes and possibly in multivesicular endosomes. Minimal colocalization was observed between TLR2 and the ER marker Calnexin (not shown), showing that TLR2 is not retained in the ER, which is in contrast to other TLRs such as TLR3 and TLR9 [53
, 54
].
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Figure 2. TLR2 is expressed in the plasma membrane, endosomes, lysosomes, and Rab-11-positive compartments but not in the Golgi of monocytes. Confocal images of freshly isolated monocytes stained intracellularly with the TLR2 mAb TL2.1 (red) and antibodies against (A) early endosome marker Eea-1 (green), (B) lysosome marker LAMP-1 (green), (C) trans-Golgi marker Golgin-97 (green), or (D) Rab-11A (green). Areas of colocalization are shown in yellow. Percent values (A–D) denote the area percentage of each marker that colocalizes with TLR2 staining. Panels to the right of each image show enlargements of two sections, denoted by squares in each image. Colocalization maps showing colocalization events between TLR2 and respective marker stainings are shown in the top panels (white). Single tracks of the respective marker (green) and TLR2 (red) are shown in the middle and bottom panels. Profile graphs show fluorescence intensity of each color in a cross-section denoted by an arrow in each image (A–D). Images of cells shown are representative of the cells observed in each dish and are representative of three experiments.
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Figure 3. Monocytes efficiently bind and internalize LTA and up-regulate TLR2. (A) Monocytes efficiently bind and internalize LTA. A+ buffy coat from healthy donors was incubated with LTARhodamine Green for 45 min at 4°C or at 37°C, 8% CO2. RBCs were subsequently lysed, and remaining cells were analyzed by flow cytometry to determine LTA binding and uptake. Populations were gated by size and granularity and CD14 high expression (monocytes), CD14 low expression (granulocytes), and CD3 or CD19 expression (lymphocytes). (B) Monocytes were fixed and stained extracellularly with antibody against TLR2, CD14, and CD36 for 45 min on ice and analyzed by flow cytometry. Monocytes were stimulated with LTA (0, 0.1, 1, 10, 100, or 1000 ng/ml) for 16 h and were subsequently stained for surface expression of (C) TLR2 or (D) CD36 prior to determination of median fluorescence by flow cytometry. Results shown are representative of three independent experiments.
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Figure 4. LTA is rapidly internalized in tubular structures and targeted to the trans-Golgi network and the ER. (A) Internalization of LTArhodamine (20 µg/ml; red) in live monocytes after 20 min of incubation at 37°C. (B–E) Monocytes incubated with LTArhodamine (red; 20 µg/ml) for 1 h at 37°C, 8% CO2, and subsequently fixed and stained intracellularly with antibodies against (B) Golgin-97 (green), (C) Eea-1 (green), (D) LAMP-1 (green), or (E) ER marker Calnexin and secondary antibody goat anti-mouseA647 (green). (F) Confocal images of live HEK293-TLR2 cells transiently expressing CFP fused to the targeting sequence of calreticulin (ERCFP), which localizes to the ER (green), incubated with LTArhodamine (red; 20 µg/ml) for 1 h at 37°C. (B–F) Percent values denote percent area of each marker that colocalizes with LTArhodamine. Panels to the right of each image (B–F) show enlargements of sections denoted by squares in each image. Colocalization maps showing colocalization events between LTArhodamine and respective marker stainings are shown in the top panels (white). Single tracks of markers are shown in green and LTArhodamine in red. Profile graphs are included, showing fluorescence intensity of each color in a cross-section denoted by an arrow in images (A–E). Images of cells shown are representative of the cells observed in each dish and are representative of three independent experiments.
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B activation in response to LTAB activation in response to LTA was only observed upon expression of TLR2 (Fig. 5
). Coexpression of TLR2 with CD36 or CD14 enhanced LTA-induced NF-B activation approximately threefold compared with cells expressing TLR2 alone (Fig. 5)
. These results indicate that CD36 and CD14 function as coreceptors for TLR2 in response to LTA and that expression of either coreceptor enhances LTA-induced NF-B activation markedly. A small, additive effect on NF-B activation was observed upon coexpression of CD36 and CD14 with TLR2 in response to LTA (Fig. 5)
. The transfection efficiency and cell viability in all the samples were similar as determined by cotransfection with a constitutively expressed Renilla luciferase vector (not shown). NF-B activation was not observed in HEK293 cells transfected with TLR4, CD14, and MD2 in response to LTA, although these cells responded normally to LPS (Fig. 5)
, illustrating the purity of the LTA.
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Figure 5. CD14 and CD36 enhance LTA-induced NF-B activation mediated by TLR2. (A) HEK293 cells transfected with a NF-B luciferase reporter plasmid, and TLR2 or TLR2 in combination with CD36 and/or CD14 for 24 h was stimulated with LTA (5 µg/ml) or LPS (100 ng/ml) for 5 h at 37°C, 8% CO2. Cells were subsequently lysed and assayed for NF-B activation. Results shown are representative of three independent experiments.
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Figure 6. CD36 and CD14 are expressed at the plasma membrane, where they colocalize with TLR2. Freshly isolated monocytes incubated with medium (A and C) or LTArhodamine for 1 h (B and D) at 37°C, 5% CO2, and subsequently fixed and stained intracellularly with TLR2 mAb TL2.1A647 (red) and anti-CD36FITC (green; A and B) or TL2.1A647 (red) and anti-CD14A488 (green; C and D). Staining was observed by confocal microscopy. Percent values shown in images denote the percentage area of CD36 or CD14 that colocalizes with TLR2 staining. Panels to the right of each image show enlargements of a section, denoted by a square in each image. Areas of colocalization between CD36 (A) or CD14 (C) and TLR2 stainings are shown in the top panels (white). Single tracks of CD36 (A) or CD14 (C) are shown in green and TLR2 in red in separate panels. (B and D) Separate tracks are shown of CD36 (B) or CD14 (D) in green, TLR2 (red), and LTA (blue). (A–D) Profile graphs show fluorescence intensity of cross-sections denoted by an arrow.
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Figure 7. Blocking CD14 or CD36 impairs LTA cell association and subsequent TNF release in monocytes. (A) Monocytes were pretreated with mAb against TLR2, CD36, CD14, or control antibody (10 µg/ml) on ice (0–4°C) for 45 min before addition of LTARhodamine Green (2 µg/ml) for 45 min on ice. Cells were subsequently washed and analyzed by flow cytometry to assess LTA binding. Dotted line denotes background fluorescence of cells incubated with medium only. (B) Monocytes were pretreated with control antibody or mAb against CD36, CD14, or TLR2 or CD36, CD14, and TLR2 in combination for 45 min at room temperature before cells were stimulated with medium, LTA (10 µg/ml), Pam3CysSK4 (50 ng/ml), or LPS (20 ng/ml) for 5 h at 37°C, 5% CO2. Supernatant was harvested and analyzed for TNF by ELISA. Results shown are representative of three independent experiments.
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TLR2 signaling in response to LTA occurs mainly at the plasma membrane, independently of receptor-mediated endocytosis via Dynamin-I
As LTA colocalized with TLR2 at the plasma membrane and along the endocytic pathway, we further examined whether internalization of LTA was necessary for signaling. Monocytes were incubated on LTA-coated wells or with LTA added in solution. Immobilization of LTA on a plastic surface should allow binding but restrict internalization of the ligand. Interestingly, we found that immobilizing LTA greatly enhanced TNF release from monocytes, compared with cells that received LTA in solution (Fig. 8A
). We argue that the prominent effect on TNF release from monocytes observed upon immobilizing LTA shows that signaling predominantly occurs at the plasma membrane and does not require internalization of LTA.
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Figure 8. TLR2 signaling in response to LTA occurs at the plasma membrane and is not dependent on Dynamin-I. (A) Immobilizing LTA on a plastic surface enhances TNF release in monocytes, which were stimulated by plating cells in wells coated with LTA or PBS or stimulated by adding medium or LTA in solution. Supernatant was harvested after overnight incubation, and TNF levels were analyzed by ELISA. Results show average TNF release of duplets and are representative of three independent experiments. (B) LTA is internalized by a receptor-mediated mechanism. Confocal images of HEK-TLR2 cells transiently expressing wild-type (WT) Dynamin-I or the mutant Dynamin-I K44A and control pcDNA3 or CD14 incubated with LTArhodamine (red) or transferrinA633 (green) for 30 min at 37°C, 8% CO2, prior to imaging. The nucleuses of cells are outlined in Dynamin-I K44A-expressing cells. (C) LTA-induced NF-B activation occurs at the plasma membrane, independent of LTA uptake. HEK293-TLR2 cells were transfected with a NF-B luciferase reporter plasmid and wild-type Dynamin-I or the mutant Dynamin-I K44A in the presence of control pcDNA3 or CD14. Cells were subsequently stimulated with LTA (5 µg/ml) or medium for 6 h, 37°C, 8% CO2, before cells were lysed and assayed for NF-B activation. Results shown are representative of three experiments.
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To assess whether internalization of LTA was necessary for signaling, NF-B activation was assessed in HEK-TLR2 cells in the absence or presence of CD14 and in the presence of wild-type Dynamin-I or the mutant K44A Dynamin-I. Figure 8C
shows that introduction of CD14 strongly enhanced LTA-induced NF-B activation but that the Dynamin-I K44A mutant did not affect NF-B activation, neither in the presence nor absence of CD14. These results suggest that CD14 enhances signaling by binding LTA to the plasma membrane and that LTA internalization is not required for signaling.
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Using a TLR2-specific mAb, we found that TLR2 rapidly traffics from the plasma membrane to early endosomes in live cells that overexpressed the receptor. Early endosomes containing TLR2 subsequently matured (Fig. 1A) . We further observed TLR2 in lysosomes. Although only 20–30% of the TLR2 antibody was observed colocalizing with LysotrackerGreen after 3 h, this observation could be a result of several factors. Incubation for longer time periods or during stimulation may have shown more TLR2 in the lysosomes. The fluorochrome bound to the TL2.1 antibodies may lose fluorescence in the acidic environment of lysosomes of live cells, or the antibodies may be degraded or uncoupled from TLR2 in these compartments, resulting in the low percentage of TLR2 mAb observed in lysosomes. Although TLR2 has been reported not to be ubiquitinylated by the ubiquitin-protein ligase TRIAD3A, in contrast to TLR4 and TLR9 [59 ], other ligases, for instance, TRIAD3B, may ubiquitinylate TLR2 and target it for degradation. The TL2.1 mAb used in this study has been shown to be presented on MHC class II and induce proliferation of a mouse C-specific human CD4+ T cell clone [60 ], supporting that TLR2 traffics along the classical MHC II pathway.
TLR2 was further found to be highly expressed in a population of Eea-1-positive early endosomes and LAMP-1-positive lysosomes in human monocytes. In contrast to previous reports [36 ], we did not observe TLR2 in the Golgi network in human monocytes; however, we did observe TLR2 in the Golgi network of HEK293 cells overexpressing the receptor (data not shown). We interpret these results as characteristic of epithelial cells or an effect of overexpression of TLR2. In monocytes, TLR2 colocalized with Rab-11A-positive structures localized in close proximity to the trans-Golgi network, suggesting that TLR2 is expressed in endosomal recycling compartments. Whether Rab-11A is required for LTA internalization and signaling remains to be investigated.
TLR2 is essential for inflammatory responses toward highly purified LTA [34 ]. The high amount of TLR2 at the plasma membrane and up-regulation of the receptor in response to LTA support the notion that TLR2 signaling occurs at the plasma membrane. Localization of TLR2 in endosomes and lysosomes, however, suggests that signaling may occur in these compartments as well, as shown for TLR4 [61 ]. Signaling from endosomes/lysosomes also occurs for TLR3 [53 ] and TLR9 [54 ], although these receptors are recruited to endosomes from the ER. Using directly labeled, functional LTA, we found that LTA was rapidly internalized in characteristic tubular structures, which colocalized with markers for ER and Golgi network, showing that LTA has an uptake and trafficking pattern in phagocytic cells, which is different from other TLR ligands such as LPS, CpG, and polyinosinic:polycytidylic acid [32 , 53 , 54 , 61 ]. The colocalization of LTA with markers of the ER and the Golgi suggests that it follows a retrograde pathway, possibly resembling the trafficking pattern of the plant toxin ricin and the bacterial toxin shiga toxin [62 ], which follow a retrograde transport to the ER [63 64 65 66 ]. Although shiga toxin is shown to be internalized in clathrin-coated pits, ricin is internalized by dynamin-, clathrin-, and caveolea-independent mechanisms [62 ]. In light of the finding that LTA appears to be targeted to the Golgi and ER, it would be interesting to compare LTA internalization with ricin and shiga toxin.
Our results demonstrate that LTA was endocytosed in a Dynamin-I-dependent manner. Previous reports have suggested that LTA is internalized by a lipid raft-dependent pathway [67 68 69 ]. Colocalization between LTA and transferrin was observed during the initial minutes of endocytosis in HEK293 cells expressing TLR2 (N. J. Nilsen, unpublished data). In addition, some colocalization between LTA and cholera toxin B was seen, predominantly in the Golgi network (N. J. Nilsen, unpublished data). These results suggest that LTA may be internalized by clathrin- and caveolea-dependent pathways. Colocalization between LTA and transferrin and cholera toxin B was, however, only partial, and the internalization pattern of LTA did not mimic the uptake of LPS, fibroblast-stimulating lipopeptide-1, or Pam3CysSK4. These ligands are internalized slower than LTA and are seen in endocytic vesicles that clearly colocalize with transferrin [35 , 61 ] (N. J. Nilsen, unpublished data). Our findings suggest that LTA may use several endocytic pathways, which also have been described for CD14-mediated LPS uptake [61 , 70 ].
The role of CD36 as a coreceptor for TLR2 in response to LTA has predominantly been studied in the presence of CD14 [37
, 39
], and the contribution of each of these coreceptors has not been compared previously. In accordance with previous reports [34
, 39
], we found that CD36 and CD14 enhanced LTA-induced, TLR2-mediated NF-B activation in transfected cells. Only a minor additive effect was observed upon coexpression of both receptors in HEK-TLR2 cells. In monocytes, CD36 and CD14 mAb inhibited cell association of LTA and TNF release to a similar extent (Fig. 7)
. The results suggest that inhibiting CD14 or CD36 on monocytes prevents binding of LTA, which in turn reduces signaling and the induction of TNF. Consequently, CD36 and CD14 appear to be important in enhancing TLR2 signaling in response to LTA. CD36, however, appears to be specifically involved in TLR2/TLR6-mediated responses but not in TLR2/TLR1-mediated responses, in line with previous observations [37
]. CD14, in contrast, enhances signaling in response to LPS and Pam3CysSK4, as well as in response to LTA.
Expression of CD14 in HEK-TLR2 cells profoundly enhanced binding of LTA to the plasma membrane and NF-B activation, suggesting that this coreceptor may up-regulate signaling by accumulating LTA at the plasma membrane. Furthermore, immobilizing LTA on a plastic surface induced a high level of TNF release in monocytes, independent of internalization of the ligand. This appears to be a specific property of LTA but not LPS or Pam3CysSK4 (S. Deininger, unpublished data). Expression of Dynamin-I K44A inhibited LTA internalization in the absence and presence of CD14, suggesting that LTA is internalized by a receptor-mediated mechanism. Expression of Dynamin-I K44A had, however, no significant effect on LTA-induced NF-B activation, showing that binding of LTA to the plasma membrane is sufficient to induce signaling. This finding is in contrast to previous reports describing that CD36-mediated internalization of LTA is required for signaling [39
]. The presence of CD36 may, however, also be required for formation of the correct receptor clustering in response to LTA or for recruitment of LTA to lipid rafts, as suggested in recent reports [38
]. In summary, our results show that signaling in response to LTA occurs independent of internalization of the ligand and provide further insight into the mechanisms of LTA internalization, trafficking, and signaling through TLR2 and its coreceptors CD14 and CD36.
Received September 27, 2007; revised March 25, 2008; accepted March 28, 2008.
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