TLR1 cooperates with TLR2 on the cell surface to initiate IL-8 secretion. Control antibody (OKT8), anti-CD14 (26ic), anti-TLR1 (GD2.F4), and anti-TLR2 (TL2.1) mAbs were immobilized on sterile high protein binding polystyrene 96-well plates from 0.2 to 0.8 μg/ml concentration. After blocking and washing, 7 × 10⁵ human PBMCs were added to the antibody-coated wells in the presence of 5 μg/ml polymyxin B. After 18 h of incubation, supernatants were harvested and levels of IL-8 were measured by ELISA.

TLR2 and TLR1 mutants inhibit NFκB activation in HEK 293 cells in stimulation with araLAM and zymosan. (A) The response of HEK293-CD14 cells to araLAM and zymosan is inhibited by a single point mutated TLR2 construct, TLR2-P681H. HEK293-CD14 cells were cotransfected with 5 ng TLR2-WT DNA and increasing amounts of TLR2-P681H (from 5 to 200 ng DNA). After 6 h of stimulation with 1 μg/ml araLAM and 10 μg/ml zymosan, cells were lysed and NFκB luciferase reporter gene activity was measured. IL-1β was used as a positive control. Luciferase activity is expressed in normalized RLU as the ratio of NFκB-dependent firefly luciferase activity to NFκB-independent renilla luciferase activity. Data shown are the mean ± SD of triplicate wells. (B) The response of HEK293-CD14 cells to araLAM and zymosan is inhibited by expression of a TLR2 mutant missing the TIR domain TLR2-ΔTIR. HEK293-CD14 cells were cotransfected with 5 ng TLR2-WT DNA and increasing amounts of TLR2-ΔTIR (from 5 to 200 ng DNA). After 6 h of stimulation with 1 μg/ml araLAM and 10 μg/ml zymosan, cells were lysed and NFκB luciferase reporter gene activity was measured. IL-1β was used as a positive control. (C) The response of HEK293-CD14 cells to araLAM and zymosan is inhibited by expression of a cytoplasmic deletion mutant of TLR1, TLR1-Δcyt. HEK293-CD14 cells were cotransfected with 5 ng TLR2-WT DNA and increasing amounts of TLR1-Δcyt (from 5 to 200 ng DNA). After 6 h of stimulation with 1 μg/ml araLAM and 10 μg/ml zymosan, cells were lysed and NFκB luciferase reporter gene activity was measured. IL-1β was used as a positive control.

NFκB signal activation requires both extracellular and intracellular domains of TLR1 and TLR2. (A) HEK293-CD14 cells were cotransfected with TLR [1–2] and TLR [2–1] DNA encoding chimeric proteins or wild-type TLR2 DNA. After 6 h of stimulation with 1 μg/ml araLAM, 100 ng/ml Pam₃CSK₄, or 10 μg/ml zymosan, cells were lysed and NFκB luciferase reporter gene activity was measured. IL-1β was used as a positive control. (B) A schematic model of cotransfection of TLR [1–2] and TLR [2–1] chimeric proteins. Co-transfection of both chimeric proteins confers responsiveness as a result of concomitant expression of both intracellular and extracellular domains of TLR1 and TLR2. With TLR1 endogenously present, transfection with TLR2-WT alone is sufficient to confer responsiveness.

Anti-TLR1 and -TLR2 mAbs block the IL-6 response of human PBMCs to araLAM and Pam₃CSK₄. Fresh human PBMCs were preincubated for 30 min with anti-TLR1 mAb (GD2.F4) or anti-TLR2 mAb (11G7) before adding the stimulants. After 18 h of stimulation with 1 μg/ml araLAM, 100 ng/ml Pam₃CSK₄ (A), 10 μg/ml zymosan, or 10 ng/ml phenol LPS (B), supernatants were harvested and IL-6 was measured by ELISA.

Confocal imaging of live HEK cells expressing fluorescent protein–tagged TLR1, TLR2, and TLR4. (A) Confocal microscopy of HEK cells stably expressing TLR1^YFP, TLR2^YFP, or TLR4^YFP. HEK cells stably expressing either TLR1^YFP, TLR2^YFP, or TLR4^YFP were grown on glass-bottom tissue culture dishes, and living cells were analyzed by confocal microscopy at 37°C without any manipulation. Shown are confocal sections in representative cells. (B) TLR1 colocalizes with TLR2 in resting HEK cells stably expressing both TLR1^YFP and TLR2^CFP. HEK cells stably expressing both TLR1^YFP and TLR2^CFP were grown on glass-bottom tissue culture dishes, and living cells were analyzed as described in A. Left, Localization of TLR2; center, distribution of TLR1; right, overlay of the left and middle images. In these cells without any manipulation and without ligand stimulation, TLR1 colocalizes with TLR2 (arrows and arrowheads). Representative confocal sections of cells are shown. (C) Surface cross-linking of TLR2^CFP by antibody leads to coaggregation of TLR1^YFP on the cell surface. Double transfectants of TLR2^CFP and TLR1^YFP were surface stained with anti-TLR2 antibody (clone TL2.1), and further cross-linked with Alexa^® 647–conjugated polyclonal rabbit anti–mouse antibody (2° Ab^Alexa674, shown in blue) on ice. After incubation for 10 min at 37°C to induce capping, living cells were analyzed by confocal microscopy, and representative cells are shown. Left, distribution (green) and surface labeling of TLR2^CFP with anti-TLR2 + 2° Ab^Alexa674 (blue); middle, TLR1^YFP distribution (red) and the overlay image of all three colors. The transmitted light image and the fluorescence intensities of a representative section of the overlay image is shown in the right panels. Surface cross-linking of TLR2^CFP by antibody (blue) induced coaggregation of TLR2^CFP and TLR1^YFP. (D) Antibody-induced capping of MHC I does not induce coaggregation with TLR2^CFP or TLR1^YFP. HEK cells stably expressing TLR2^CFP and TLR1^YFP were stained for MHC I using a mouse monoclonal anti-HLA I antibody, counterstained and processed as described in B. Distribution of TLR2^CFP (green) and surface labeling of MHC class I with anti-MHC I + 2° Ab^Alexa674 (blue) are shown in the left panels; the middle panels demonstrate TLR1^YFP localization (red) and the overlay of all three colors. The transmitted light image and fluorescence intensities of a representative section of the overlay image are displayed in the right panels. MHC I cross-linking (blue) does not co-patch either TLR2^CFP or TLR1^YFP. (E) Antibody-induced capping of TLR2 does not coaggregate TLR4. Stably transfected HEK-TLR4^YFP cells (green) were transiently transfected with TLR2. Antibody-induced capping of TLR2 was induced by sequentially incubating the cells with anti-TLR2 (clone TL2.1) and anti-mouse IgG (Alexa^® 647 conjugated; red). Aggregation of TLR2 did not influence the expression pattern of TLR4.

Neither the intracellular nor the extracellular domain of TLR2 is sufficient to confer NFκB signal activation. (A) HEK293-CD14 cells were transfected with either TLR [1–2] or TLR [2–1] DNA encoding chimeric protein and wild-type TLR2 DNA. After 6 h of stimulation with 1 μg/ml araLAM, 100 ng/ml Pam₃CSK₄, or 10 μg/ml zymosan, cells were lysed and NFκB luciferase reporter gene activity was measured. IL-1β was used as a positive control. (B) A schematic model of separate TLR1 and TLR2 chimeric protein transfection. With TLR1 endogenously expressed, transfection only of the intracellular portion of TLR2 (TLR [1–2]) does not confer responsiveness as a result of missing TLR2 extracellular domain causing ligand recognition failure. Transfection with only the extracellular portion of TLR2 (TLR [2–1]) is not sufficient to confer responsiveness because the TLR1 intracellular domain lacks the TLR2 intracellular domain for effective initiation of signaling pathways. With TLR1 endogenously present, transfection of TLR2-WT protein confers responsiveness by providing both extra- and intracellular domains needed for ligand recognition and signal activation respectively.

Heterologous expression of the extracellular domains of TLR1 and TLR2 together with heterologous expression of the TIR domains of TLR1 and TLR2 is sufficient for NFκB signal activation. (A) HEK293-CD14 cells were cotransfected with TLR [1–2] and TLR [2–1 TIR] DNA encoding chimeric proteins or wild-type TLR2 DNA. After 6 h of stimulation with 1 μg/ml araLAM, 100 ng/ml Pam₃CSK₄, or 10 μg/ml zymosan, cells were lysed and NFκB luciferase reporter gene activity was measured. IL-1β was used as a positive control. (B) A schematic model of cotransfection of TLR [1–2] and TLR [2–1 TIR] chimeric proteins. Co-transfection of TLR [1–2] chimeric protein containing the entire intracellular domain of TLR2 (including the TIR domain) with the TLR [2–1 TIR] chimeric protein containing the TIR domain of TLR1, confers responsiveness. The transfected cells express the extracellular domains of TLR1 and TLR2 (ligand recognition) as well as both the TIR domains of TLR1 and TLR2 (signal transduction). With TLR1 endogenously present, transfection with TLR2-WT alone is sufficient to confer responsiveness.