Journal:

Article Title: Distribution of Toll-like receptor 1 and Toll-like receptor 2 in human lymphoid tissue

doi: 10.1046/j.1365-2567.2003.01563.x

Figure Lengend Snippet: Antibodies used to evaluate Toll-like receptor 1 (TLR1) and Toll-like receptor 2 (TLR2) expression in human lymphoid tissue

Article Snippet: Western blot analysis and cell transfection studies demonstrated that the TLR1 16 and TLR2 (see ref. 9 for 2392 and company insert for TLR2.3) antibodies used in this study exhibited no cross-reactivity against the other TLR protein. table ft1 table-wrap mode="anchored" t5 caption a7 Antibody specificity Clone Isotype Source CD3 B355.1 IgG3 Biomeda CD20 L-26 IgG2a Dako CD14 RPA-M1 IgG2b Zymed CD68 Y1/82 A IgG2b Phamingen CD1a NA1/34 IgG2a Dako CD1b BCD1b3.1 IgG1 Ref. 37 TLR1 GD2.F4 IgG1 Alexis; ref. 14 TLR2 2392 IgG1 Ref. 7 TLR2 TLR2.3 IgG2a Alexis Cytokeratin 8 35BH11 IgM Dako Cytokeratin 10/13 DE-K13 IgG2a Santa Cruz Biotechnology Cytokeratin 14 CKB1 IgM Sigma Factor VIII Polyclonal Rabbit Zymed Open in a separate window Antibodies used to evaluate Toll-like receptor 1 (TLR1) and Toll-like receptor 2 (TLR2) expression in human lymphoid tissue

Techniques: Expressing

Journal:

Article Title: Distribution of Toll-like receptor 1 and Toll-like receptor 2 in human lymphoid tissue

doi: 10.1046/j.1365-2567.2003.01563.x

Figure Lengend Snippet: Toll-like receptor 2 (TLR2) expression in human lymphoid tissue. Lymphoid tissue was labelled with monoclonal antibodies (mAbs) specific for TLR2, CD14 and CD1a using the immunoperoxidase method. Original magnification 100× (panels a, c and d); 400× (b). The box in panel (a) indicates the section magnified in panel (b). (A) Human lymph nodes labelled with mAbs specific for TLR2+ cells are located in the medullary cords (M) of the lymph nodes. Macrophages (CD14+) and dendritic cells (DCs) (CD1a+) localized to the same region as TLR2-positive cells TLR2 expression was weak to negative in follicles (F). (B) TLR2-expressing cells in human spleen are located predominantly at the splenic cords and at the interface between the white pulp (WP) and the red pulp (RP) or marginal zone (MZ), the same site as macrophages and DCs. (C) TLR2-positive cells in thymus are found predominantly at the medullary junction (MJ) with a limited number located at the cortex (C) and the medulla (M). Macrophages and DCs are scattered throughout the medulla, cortex and medullary junction. CD1a+ thymocytes are abundant in the cortex. (D) TLR2-positive cells in tonsil are localized in the medullary area, as are macrophages and DCs.

Article Snippet: Western blot analysis and cell transfection studies demonstrated that the TLR1 16 and TLR2 (see ref. 9 for 2392 and company insert for TLR2.3) antibodies used in this study exhibited no cross-reactivity against the other TLR protein. table ft1 table-wrap mode="anchored" t5 caption a7 Antibody specificity Clone Isotype Source CD3 B355.1 IgG3 Biomeda CD20 L-26 IgG2a Dako CD14 RPA-M1 IgG2b Zymed CD68 Y1/82 A IgG2b Phamingen CD1a NA1/34 IgG2a Dako CD1b BCD1b3.1 IgG1 Ref. 37 TLR1 GD2.F4 IgG1 Alexis; ref. 14 TLR2 2392 IgG1 Ref. 7 TLR2 TLR2.3 IgG2a Alexis Cytokeratin 8 35BH11 IgM Dako Cytokeratin 10/13 DE-K13 IgG2a Santa Cruz Biotechnology Cytokeratin 14 CKB1 IgM Sigma Factor VIII Polyclonal Rabbit Zymed Open in a separate window Antibodies used to evaluate Toll-like receptor 1 (TLR1) and Toll-like receptor 2 (TLR2) expression in human lymphoid tissue

Techniques: Expressing, Bioprocessing

Journal:

Article Title: Distribution of Toll-like receptor 1 and Toll-like receptor 2 in human lymphoid tissue

doi: 10.1046/j.1365-2567.2003.01563.x

Figure Lengend Snippet: Toll-like receptor 2 (TLR2) expression on monocytes and dendritic cells (DCs) in human lymphoid tissue. TLR2-expressing cells were characterized for cell lineage in human tonsil using two-colour immunofluorescence confocal laser microscopy. TLR2-positive cells expressed markers for DCs (CD1a) and macrophages (CD68 and CD14), but did not express T-cell (CD3) or B-cell (CD20) markers. Green images (left column) are CD markers, red images (center column) are TLR2 and superimposed images are shown in right column. Magnification 630×. The frequency of TLR2+ cells in lymphoid tissue is greater than the number of macrophages or DCs in situ as both macrophages and DCs populate lymphoid tissues and therefore the number of TLR2+ cells should be no less than the sum of CD14+ and CD1a+ cells.

Article Snippet: Western blot analysis and cell transfection studies demonstrated that the TLR1 16 and TLR2 (see ref. 9 for 2392 and company insert for TLR2.3) antibodies used in this study exhibited no cross-reactivity against the other TLR protein. table ft1 table-wrap mode="anchored" t5 caption a7 Antibody specificity Clone Isotype Source CD3 B355.1 IgG3 Biomeda CD20 L-26 IgG2a Dako CD14 RPA-M1 IgG2b Zymed CD68 Y1/82 A IgG2b Phamingen CD1a NA1/34 IgG2a Dako CD1b BCD1b3.1 IgG1 Ref. 37 TLR1 GD2.F4 IgG1 Alexis; ref. 14 TLR2 2392 IgG1 Ref. 7 TLR2 TLR2.3 IgG2a Alexis Cytokeratin 8 35BH11 IgM Dako Cytokeratin 10/13 DE-K13 IgG2a Santa Cruz Biotechnology Cytokeratin 14 CKB1 IgM Sigma Factor VIII Polyclonal Rabbit Zymed Open in a separate window Antibodies used to evaluate Toll-like receptor 1 (TLR1) and Toll-like receptor 2 (TLR2) expression in human lymphoid tissue

Techniques: Expressing, Immunofluorescence, Microscopy, In Situ

Journal:

Article Title: Distribution of Toll-like receptor 1 and Toll-like receptor 2 in human lymphoid tissue

doi: 10.1046/j.1365-2567.2003.01563.x

Figure Lengend Snippet: Toll-like receptor 1 (TLR1) expression on monocytes and dendritic cells (DCs) in lymphoid tissue. (A) Human tonsils labelled with monoclonal antibodies (mAbs) specific for TLR1 (a, b). TLR1-positive cells in tonsil localized to the medulla. Original magnification 200× (a) and 400× (b). The box in panel (a) indicates the region magnified in panel (b). (B) TLR1-expressing cells were characterized for cell lineage in human tonsil using two-colour immunofluorescence confocal laser microscopy. TLR1-positive cells expressed markers for DCs (CD1a) and macrophages (CD14), but did not express T-cell (CD3) or B-cell (CD20) markers. Green images (left column) are CD markers, red images (centre column) are TLR1 and superimposed images are shown in the right column. Original magnification 630×. An arrow denotes the CD14+ cell in the center of the panel. (C) TLR1 co-localizes with TLR2 in lymphoid tissue. TLR1 (green) and TLR2 (red) were labelled and detected as described above (Fig. 2) and superimposed to identify co-localization.

Article Snippet: Western blot analysis and cell transfection studies demonstrated that the TLR1 16 and TLR2 (see ref. 9 for 2392 and company insert for TLR2.3) antibodies used in this study exhibited no cross-reactivity against the other TLR protein. table ft1 table-wrap mode="anchored" t5 caption a7 Antibody specificity Clone Isotype Source CD3 B355.1 IgG3 Biomeda CD20 L-26 IgG2a Dako CD14 RPA-M1 IgG2b Zymed CD68 Y1/82 A IgG2b Phamingen CD1a NA1/34 IgG2a Dako CD1b BCD1b3.1 IgG1 Ref. 37 TLR1 GD2.F4 IgG1 Alexis; ref. 14 TLR2 2392 IgG1 Ref. 7 TLR2 TLR2.3 IgG2a Alexis Cytokeratin 8 35BH11 IgM Dako Cytokeratin 10/13 DE-K13 IgG2a Santa Cruz Biotechnology Cytokeratin 14 CKB1 IgM Sigma Factor VIII Polyclonal Rabbit Zymed Open in a separate window Antibodies used to evaluate Toll-like receptor 1 (TLR1) and Toll-like receptor 2 (TLR2) expression in human lymphoid tissue

Techniques: Expressing, Bioprocessing, Immunofluorescence, Microscopy

Journal:

Article Title: Distribution of Toll-like receptor 1 and Toll-like receptor 2 in human lymphoid tissue

doi: 10.1046/j.1365-2567.2003.01563.x

Figure Lengend Snippet: Toll-like receptor 2 (TLR2) expression on epithelial cells in lymphoid tissue. (A) Immunoperoxidase staining of sections demonstrates that TLR2-positive cells are also distributed throughout the tonsillar crypt epithelium (TCE). Original magnification 100× (a) and 200× (b). (B) Double-immunofluorescence labelling of tonsil sections with cytokeratin (CK) 8, 14 and 10/13 (green; left panel of each row) and TLR2 (red; centre panel of each row) demonstrate co-localization of TLR2 with CK10/13, but not CK8 or CK14. Original magnification 630×. (C) Double-immunofluorescence labelling with endothelial cell marker Factor VIII (green; first panel) and TLR2 (red) illustrates a lack of co-localization (right panel).

Article Snippet: Western blot analysis and cell transfection studies demonstrated that the TLR1 16 and TLR2 (see ref. 9 for 2392 and company insert for TLR2.3) antibodies used in this study exhibited no cross-reactivity against the other TLR protein. table ft1 table-wrap mode="anchored" t5 caption a7 Antibody specificity Clone Isotype Source CD3 B355.1 IgG3 Biomeda CD20 L-26 IgG2a Dako CD14 RPA-M1 IgG2b Zymed CD68 Y1/82 A IgG2b Phamingen CD1a NA1/34 IgG2a Dako CD1b BCD1b3.1 IgG1 Ref. 37 TLR1 GD2.F4 IgG1 Alexis; ref. 14 TLR2 2392 IgG1 Ref. 7 TLR2 TLR2.3 IgG2a Alexis Cytokeratin 8 35BH11 IgM Dako Cytokeratin 10/13 DE-K13 IgG2a Santa Cruz Biotechnology Cytokeratin 14 CKB1 IgM Sigma Factor VIII Polyclonal Rabbit Zymed Open in a separate window Antibodies used to evaluate Toll-like receptor 1 (TLR1) and Toll-like receptor 2 (TLR2) expression in human lymphoid tissue

Techniques: Expressing, Immunoperoxidase Staining, Immunofluorescence, Marker

Journal:

Article Title: TLR2 is mobilized into an apical lipid raft receptor complex to signal infection in airway epithelial cells

doi: 10.1172/JCI200420773

Figure Lengend Snippet: Distribution of asialoGM1, TLR2, and TLR4 in infected airway cells. (A) Confocal microscopy was used to image monolayers of polarized 16HBE cells transfected with Flag epitope–tagged TLR2 or TLR4 and stimulated with P. aeruginosa PAO1. z-sections treated with anti-Flag labeled with TRITC (red) and with anti-asialoGM1 labeled with FITC (green) are shown. AsialoGM1 (aGM1) is apical and colocalizes with TLR2 (yellow) in discrete clusters along the apical surface of the monolayers. TLR4 is more diffuse, and colocalization with asialoGM1 is not appreciable. (B) Human airway cells in primary culture isolated from nasal polyps from a CF patient and stimulated with P. aeruginosa PAO1 were stained for asialoGM1 labeled with TRITC and TLR2 labeled with FITC and show abundant colocalization of these receptors.

Article Snippet: Rabbit polyclonal antibodies used were anti–caveolin-1, anti-TLR2, anti–IRAK-1, anti-MYD88, anti-TRAF6, and anti-TLR4 (Santa Cruz Biotechnology Inc., Santa Cruz, California, USA); anti-TLR2 antisera from Tularik (South San Francisco, California, USA); and anti-asialoGM1 (Wako, Richmond, Virginia, USA).

Techniques: Infection, Confocal Microscopy, Transfection, FLAG-tag, Labeling, Isolation, Staining

Journal:

Article Title: TLR2 is mobilized into an apical lipid raft receptor complex to signal infection in airway epithelial cells

doi: 10.1172/JCI200420773

Figure Lengend Snippet: Identification of apically exposed components on polarized 16HBE cells exposed to bacteria. (A) Surface-exposed components of the airway cells were biotinylated under control conditions (–) and after a 1-hour exposure to S. aureus (+). After immunoprecipitation with streptavidin, Western hybridizations were done and asialoGM1, TLR2, and caveolin-1 (Cav-1), as well as the kinases IRAK-1 and TRAF6, were detected. (B) Coimmunoprecipitation studies demonstrate TLR2 but not TLR4 in a receptor complex along with asialoGM1. Coimmunoprecipitations of whole-cell lysates from control and S. aureus–stimulated cells were done using anti–caveolin-1, anti-TLR2, and anti-asialoGM1 as capture antibodies with screening for expected components of the TLR pathway, MyD88 and IRAK-1, as well as c-Src and TLR4.

Article Snippet: Rabbit polyclonal antibodies used were anti–caveolin-1, anti-TLR2, anti–IRAK-1, anti-MYD88, anti-TRAF6, and anti-TLR4 (Santa Cruz Biotechnology Inc., Santa Cruz, California, USA); anti-TLR2 antisera from Tularik (South San Francisco, California, USA); and anti-asialoGM1 (Wako, Richmond, Virginia, USA).

Techniques: Immunoprecipitation, Western Blot

Journal:

Article Title: TLR2 is mobilized into an apical lipid raft receptor complex to signal infection in airway epithelial cells

doi: 10.1172/JCI200420773

Figure Lengend Snippet: Surface colocalization of MyD88, IRAK-1, and TRAF6 with TLR2 confirmed by confocal microscopy. After stimulation with S. aureus, permeabilized cells were stained with the kinases, each labeled with an Alexa Fluor 488–tagged secondary antibody (green). All were found at the cell surface, colocalized (yellow) with TLR2, labeled with an Alexa Fluor 594–tagged secondary antibody (red).

Article Snippet: Rabbit polyclonal antibodies used were anti–caveolin-1, anti-TLR2, anti–IRAK-1, anti-MYD88, anti-TRAF6, and anti-TLR4 (Santa Cruz Biotechnology Inc., Santa Cruz, California, USA); anti-TLR2 antisera from Tularik (South San Francisco, California, USA); and anti-asialoGM1 (Wako, Richmond, Virginia, USA).

Techniques: Confocal Microscopy, Staining, Labeling

Journal:

Article Title: TLR2 is mobilized into an apical lipid raft receptor complex to signal infection in airway epithelial cells

doi: 10.1172/JCI200420773

Figure Lengend Snippet: Lipid rafts are involved in clustering of receptors and signaling. (A) Confocal z-section images demonstrate caveolin-1 labeled with Alexa Fluor 594 (red) and GM1, identified with cholera toxin β-subunit (CTB) conjugated to Alexa Fluor 488 (green), on the apical surfaces of polarized 16HBE cells permeabilized after stimulation with Pam3Cys-Ser-Lys4. (B) CF nasal polyp cells were infected with P. aeruginosa PAO1 (PA) and grown on semipermeable supports, and transmission electron micrograph were obtained after 3 hours of bacterial exposure. Arrow (Cav) indicates the flask-shaped electron-dense structures typical of caveolae (magnification, ∞30,000). (C) Flow cytometry was used to detect superficial caveolin-1 on polarized 16HBE cells after exposure to S. aureus. Unstim, unstimulated. (D) Aliquots of Triton-insoluble lysates of 16HBE cells obtained before (–) and after (+) stimulation with P. aeruginosa PAO1 were fractionated on discontinuous sucrose gradients (4–40%) and were immunoblotted with anti-flotillin, anti-caveolin, anti-TLR-2, anti-IRAK-1, or anti-asialoGM1. Downward arrow indicates raft fraction containing all the components after stimulation. (E) Aliquots of sucrose gradient fractions from cells treated with filipin prior to stimulation with P. aeruginosa PAO1 were immunoblotted with anti-flotillin, anti-TLR2, and anti-asialoGM1.

Article Snippet: Rabbit polyclonal antibodies used were anti–caveolin-1, anti-TLR2, anti–IRAK-1, anti-MYD88, anti-TRAF6, and anti-TLR4 (Santa Cruz Biotechnology Inc., Santa Cruz, California, USA); anti-TLR2 antisera from Tularik (South San Francisco, California, USA); and anti-asialoGM1 (Wako, Richmond, Virginia, USA).

Techniques: Labeling, Infection, Transmission Assay, Flow Cytometry

Journal:

Article Title: TLR2 is mobilized into an apical lipid raft receptor complex to signal infection in airway epithelial cells

doi: 10.1172/JCI200420773

Figure Lengend Snippet: Mobilization of TLR2 and asialoGM1 in response to bacteria or bacterial ligands. Flow cytometry was used to quantify exposed asialoGM1, TLR2, and TLR4 on primary (HNP) cells (A) or 16HBE cells (B) after stimulation with P. aeruginosa or S. aureus or with monoclonal anti-asialoGM1, Pam3Cys-Ser-Lys4 (Pam3Cys), a TLR2 ligand, or LPS, a TLR4 ligand. Peaks outlined with a thin black line indicate binding by secondary antibody alone; gray-shaded peaks represent the labeled population under control conditions; and peaks demarcated by the heavy black line represent the population after stimulation.

Article Snippet: Rabbit polyclonal antibodies used were anti–caveolin-1, anti-TLR2, anti–IRAK-1, anti-MYD88, anti-TRAF6, and anti-TLR4 (Santa Cruz Biotechnology Inc., Santa Cruz, California, USA); anti-TLR2 antisera from Tularik (South San Francisco, California, USA); and anti-asialoGM1 (Wako, Richmond, Virginia, USA).

Techniques: Flow Cytometry, Binding Assay, Labeling

Journal:

Article Title: TLR2 is mobilized into an apical lipid raft receptor complex to signal infection in airway epithelial cells

doi: 10.1172/JCI200420773

Figure Lengend Snippet: Activation of NF-κB and IL-8 by bacteria or bacterial agonists is inhibited by DN mutations in TLR2 and MyD88. (A) IL-8 expression in 1HAEo- cells transfected with plasmids containing wild-type, DN TLR2, or a vector control was quantified by ELISA after exposure to P. aeruginosa PAO1, S. aureus RN6390, or anti-asialoGM1. Values represent the fold increase in IL-8 compared with that of unstimulated cells. IL-8 in cells stimulated by PAO1 was 1.9–2.5 ng/ml (*P < 0.001). (B) NF-κB luciferase activity in 1HAEo- cells transfected with plasmids expressing TLR2 DN or MyD88 DN constructs compared with that of cells transfected with the corresponding empty vector control was significantly inhibited after stimulation with P. aeruginosa PAO1, S. aureus RN6390, anti-asialoGM1, or Pam3Cys-Ser-Lys4 (*P < 0.001 for each). The TLR4 DN construct did not inhibit NF-κB luciferase activity. NF-κB luciferase activity for cells expressing the control vector was normalized for each stimulus and represents three- to fivefold increases over that of unstimulated cells. (C) Inhibition of IL-8 activation in the presence of filipin. IL-8 production induced by P. aeruginosa, S. aureus, anti-asialoGM1 (aGM1), or Pam3Cys-Ser-Lys4, but not TNF-α, was significantly reduced by filipin (P < 0.05, P < 0.001, P < 0.05, and P > 0.05, respectively).

Article Snippet: Rabbit polyclonal antibodies used were anti–caveolin-1, anti-TLR2, anti–IRAK-1, anti-MYD88, anti-TRAF6, and anti-TLR4 (Santa Cruz Biotechnology Inc., Santa Cruz, California, USA); anti-TLR2 antisera from Tularik (South San Francisco, California, USA); and anti-asialoGM1 (Wako, Richmond, Virginia, USA).

Techniques: Activation Assay, Expressing, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Luciferase, Activity Assay, Construct, Inhibition