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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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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.
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fig1: 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.

Mentions: Genetic studies suggest that TLR2 is required for the recognition of a diverse group of microbial ligands, including araLAM, Pam3CSK4, and zymosan. The role of TLR1 and TLR2 in the response of normal human PBMCs to these ligands was analyzed in antibody-blocking experiments. Pretreatment of PBMCs with either anti-TLR1 or -TLR2 (11G7) antibodies blocked IL-6 cytokine production in response to araLAM and Pam3CSK4 (Fig. 1 A). In contrast, both antibodies failed to inhibit the IL-6 cytokine secretion after stimulation with zymosan. Moreover, as expected, addition of anti-TLR1 or -TLR2 antibodies to PBMCs did not exert any blocking effect to LPS stimulation (Fig. 1 B). These results suggest that TLR1 and TLR2 both participate in the response to araLAM and Pam3CSK4.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: 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.
Mentions: Genetic studies suggest that TLR2 is required for the recognition of a diverse group of microbial ligands, including araLAM, Pam3CSK4, and zymosan. The role of TLR1 and TLR2 in the response of normal human PBMCs to these ligands was analyzed in antibody-blocking experiments. Pretreatment of PBMCs with either anti-TLR1 or -TLR2 (11G7) antibodies blocked IL-6 cytokine production in response to araLAM and Pam3CSK4 (Fig. 1 A). In contrast, both antibodies failed to inhibit the IL-6 cytokine secretion after stimulation with zymosan. Moreover, as expected, addition of anti-TLR1 or -TLR2 antibodies to PBMCs did not exert any blocking effect to LPS stimulation (Fig. 1 B). These results suggest that TLR1 and TLR2 both participate in the response to araLAM and Pam3CSK4.

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