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Importance of extra- and intracellular domains of TLR1 and TLR2 in NFkappa B signaling.

Sandor F, Latz E, Re F, Mandell L, Repik G, Golenbock DT, Espevik T, Kurt-Jones EA, Finberg RW - J. Cell Biol. (2003)

Bottom Line: Simultaneous cross-linking of both receptors resulted in ligand-independent signal transduction.Using chimeric TLRs, we found that expression of the extracellular domains along with simultaneous expression of the intracellular domains of both TLRs was necessary to achieve functional signaling.The domains from each receptor did not need to be contained within a single contiguous protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Massachusetts Medical Center, Worcester, MA 01605-2324, USA.

ABSTRACT
Recognition of ligands by toll-like receptor (TLR) 2 requires interactions with other TLRs. TLRs form a combinatorial repertoire to discriminate between the diverse microbial ligands. Diversity results from extracellular and intracellular interactions of different TLRs. This paper demonstrates that TLR1 and TLR2 are required for ara-lipoarabinomannan- and tripalmitoyl cysteinyl lipopeptide-stimulated cytokine secretion from mononuclear cells. Confocal microscopy revealed that TLR1 and TLR2 cotranslationally form heterodimeric complexes on the cell surface and in the cytosol. Simultaneous cross-linking of both receptors resulted in ligand-independent signal transduction. Using chimeric TLRs, we found that expression of the extracellular domains along with simultaneous expression of the intracellular domains of both TLRs was necessary to achieve functional signaling. The domains from each receptor did not need to be contained within a single contiguous protein. Chimeric TLR analysis further defined the toll/IL-1R domains as the area of crucial intracellular TLR1-TLR2 interaction.

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Related in: MedlinePlus

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.
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fig2: 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.

Mentions: Both antibody-blocking experiments and TLR knockout animal analyses suggest that TLR2 signaling involves cooperation with other TLRs, particularly TLR1 and TLR6. Thus, a functional signal transduction complex seems to require elements of both receptors. We hypothesized that cross-linking TLR1 and TLR2 might mimic their engagement by a ligand and thus activate signal transduction and cytokine secretion. We analyzed the ability of plate-bound antibodies to TLRs to activate normal human cells. PBMCs were incubated on sterile tissue culture plates coated with mAbs to TLR1 (GD2.F4), TLR2 (2.1) alone, or in combination. We observed that a combination of anti-TLR1 and -TLR2 specific mAbs activated IL-8 secretion from PBMCs in a dose-dependent manner. It should be noted that the aggregation of TLRs by soluble antibodies did not activate these cells to secrete cytokines, whereas the same anti-TLR antibodies did activate cytokine secretion when they were prebound to tissue culture plates upon which the TLR-expressing cells were cultured. Individually, neither anti-TLR1, anti-TLR2, nor isotype control antibodies alone were sufficient to elicit IL-8 secretion (Fig. 2).


Importance of extra- and intracellular domains of TLR1 and TLR2 in NFkappa B signaling.

Sandor F, Latz E, Re F, Mandell L, Repik G, Golenbock DT, Espevik T, Kurt-Jones EA, Finberg RW - J. Cell Biol. (2003)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172862&req=5

fig2: 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.
Mentions: Both antibody-blocking experiments and TLR knockout animal analyses suggest that TLR2 signaling involves cooperation with other TLRs, particularly TLR1 and TLR6. Thus, a functional signal transduction complex seems to require elements of both receptors. We hypothesized that cross-linking TLR1 and TLR2 might mimic their engagement by a ligand and thus activate signal transduction and cytokine secretion. We analyzed the ability of plate-bound antibodies to TLRs to activate normal human cells. PBMCs were incubated on sterile tissue culture plates coated with mAbs to TLR1 (GD2.F4), TLR2 (2.1) alone, or in combination. We observed that a combination of anti-TLR1 and -TLR2 specific mAbs activated IL-8 secretion from PBMCs in a dose-dependent manner. It should be noted that the aggregation of TLRs by soluble antibodies did not activate these cells to secrete cytokines, whereas the same anti-TLR antibodies did activate cytokine secretion when they were prebound to tissue culture plates upon which the TLR-expressing cells were cultured. Individually, neither anti-TLR1, anti-TLR2, nor isotype control antibodies alone were sufficient to elicit IL-8 secretion (Fig. 2).

Bottom Line: Simultaneous cross-linking of both receptors resulted in ligand-independent signal transduction.Using chimeric TLRs, we found that expression of the extracellular domains along with simultaneous expression of the intracellular domains of both TLRs was necessary to achieve functional signaling.The domains from each receptor did not need to be contained within a single contiguous protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Massachusetts Medical Center, Worcester, MA 01605-2324, USA.

ABSTRACT
Recognition of ligands by toll-like receptor (TLR) 2 requires interactions with other TLRs. TLRs form a combinatorial repertoire to discriminate between the diverse microbial ligands. Diversity results from extracellular and intracellular interactions of different TLRs. This paper demonstrates that TLR1 and TLR2 are required for ara-lipoarabinomannan- and tripalmitoyl cysteinyl lipopeptide-stimulated cytokine secretion from mononuclear cells. Confocal microscopy revealed that TLR1 and TLR2 cotranslationally form heterodimeric complexes on the cell surface and in the cytosol. Simultaneous cross-linking of both receptors resulted in ligand-independent signal transduction. Using chimeric TLRs, we found that expression of the extracellular domains along with simultaneous expression of the intracellular domains of both TLRs was necessary to achieve functional signaling. The domains from each receptor did not need to be contained within a single contiguous protein. Chimeric TLR analysis further defined the toll/IL-1R domains as the area of crucial intracellular TLR1-TLR2 interaction.

Show MeSH
Related in: MedlinePlus