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Oligosaccharides of Hyaluronan activate dendritic cells via toll-like receptor 4.

Termeer C, Benedix F, Sleeman J, Fieber C, Voith U, Ahrens T, Miyake K, Freudenberg M, Galanos C, Simon JC - J. Exp. Med. (2002)

Bottom Line: Western blot analysis revealed that sHA treatment resulted in distinct phosphorylation of p38/p42/44 MAP-kinases and nuclear translocation of nuclear factor (NF)-kappa B, all components of the TLR-4 signaling pathway.Finally, intravenous injection of sHA-induced DC emigration from the skin and their phenotypic and functional maturation in the spleen, again depending on the expression of TLR-4.In conclusion, this is the first report that polysaccharide degradation products of the extracellular matrix produced during inflammation might serve as an endogenous ligand for the TLR-4 complex on DCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, University of Freiburg, Freiburg D-79104, Germany. Termeer@haut.ukl.uni-freiburg.de

ABSTRACT
Low molecular weight fragmentation products of the polysaccharide of Hyaluronic acid (sHA) produced during inflammation have been shown to be potent activators of immunocompetent cells such as dendritic cells (DCs) and macrophages. Here we report that sHA induces maturation of DCs via the Toll-like receptor (TLR)-4, a receptor complex associated with innate immunity and host defense against bacterial infection. Bone marrow-derived DCs from C3H/HeJ and C57BL/10ScCr mice carrying mutant TLR-4 alleles were nonresponsive to sHA-induced phenotypic and functional maturation. Conversely, DCs from TLR-2-deficient mice were still susceptible to sHA. In accordance, addition of an anti-TLR-4 mAb to human monocyte-derived DCs blocked sHA-induced tumor necrosis factor alpha production. Western blot analysis revealed that sHA treatment resulted in distinct phosphorylation of p38/p42/44 MAP-kinases and nuclear translocation of nuclear factor (NF)-kappa B, all components of the TLR-4 signaling pathway. Blockade of this pathway by specific inhibitors completely abrogated the sHA-induced DC maturation. Finally, intravenous injection of sHA-induced DC emigration from the skin and their phenotypic and functional maturation in the spleen, again depending on the expression of TLR-4. In conclusion, this is the first report that polysaccharide degradation products of the extracellular matrix produced during inflammation might serve as an endogenous ligand for the TLR-4 complex on DCs.

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Analysis of TNF-α production by DCs in response to sHA stimulation. (A and B) Human monocyte–derived day 4 DCs were treated with 25 μg/ml sHA or 100 ng/ml LPS for the indicated times. After stimulation, (A) cells were harvested and RNA was prepared for TNF-α–specific RT-PCR, and (B) cell-free supernatants were collected and assayed by TNF-α ELISA. The dotted line shows the response to LPS, the solid line the response to sHA. Data represent the mean TNF-α release of triplicate values; pg/mg total protein ± SD. (C) DCs were incubated for 24 h with the indicated concentrations of sHA or 100 ng/ml LPS and cell-free supernatants were collected and assayed by TNF-α ELISA as detailed above.
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fig2: Analysis of TNF-α production by DCs in response to sHA stimulation. (A and B) Human monocyte–derived day 4 DCs were treated with 25 μg/ml sHA or 100 ng/ml LPS for the indicated times. After stimulation, (A) cells were harvested and RNA was prepared for TNF-α–specific RT-PCR, and (B) cell-free supernatants were collected and assayed by TNF-α ELISA. The dotted line shows the response to LPS, the solid line the response to sHA. Data represent the mean TNF-α release of triplicate values; pg/mg total protein ± SD. (C) DCs were incubated for 24 h with the indicated concentrations of sHA or 100 ng/ml LPS and cell-free supernatants were collected and assayed by TNF-α ELISA as detailed above.

Mentions: We have previously shown that DC stimulated with sHA secrete large quantities of proinflammatory cytokines, such as IL-1β, TNF-α, and IL-12, and that sHA-induced DC maturation is TNF-α dependent (5). To further characterize the time and dose dependence of sHA-induced TNF-α expression, we used RT-PCR to analyze TNF-α mRNA expression after sHA treatment of human DCs. Rapid induction of TNF-α mRNA was observed, which was detectable as early as 4 h after stimulation of DCs with either 25 μg/ml sHA or 100 ng/ml LPS (Fig. 2 A). At this dose the sHA-induced TNF-α expression was more long-lasting compared with that induced by LPS (Fig. 2 A). When the overall amount of TNF-α protein secreted into the culture medium after 24 h of stimulation with different concentrations of sHA was quantified by ELISA, there was a clear dose response in TNF-α production, with up to 4 ng/ml being produced in response to 50 μg/ml sHA (Fig. 2 B and C). The kinetics of TNF-α induction by sHA showed a late onset starting after 4–5 h of coincubation, reaching near-peak levels after 12 h, with a slight further increase up to 24 h. This effect was concentration dependent, as 10 μg/ml sHA sufficed to induce a significant TNF-α production, while saturation was observed at concentrations >20 μg/ml (Fig. 2 C). In contrast, TNF-α induction by 100 ng/ml LPS occurred much more rapidly, with significant amounts being produced after 30 min (Fig. 2 B). We conclude that the TNF-α response is a very sensitive parameter to measure the sHA-induced maturation of DC, therefore it was adopted as the major readout-system in further experiments.


Oligosaccharides of Hyaluronan activate dendritic cells via toll-like receptor 4.

Termeer C, Benedix F, Sleeman J, Fieber C, Voith U, Ahrens T, Miyake K, Freudenberg M, Galanos C, Simon JC - J. Exp. Med. (2002)

Analysis of TNF-α production by DCs in response to sHA stimulation. (A and B) Human monocyte–derived day 4 DCs were treated with 25 μg/ml sHA or 100 ng/ml LPS for the indicated times. After stimulation, (A) cells were harvested and RNA was prepared for TNF-α–specific RT-PCR, and (B) cell-free supernatants were collected and assayed by TNF-α ELISA. The dotted line shows the response to LPS, the solid line the response to sHA. Data represent the mean TNF-α release of triplicate values; pg/mg total protein ± SD. (C) DCs were incubated for 24 h with the indicated concentrations of sHA or 100 ng/ml LPS and cell-free supernatants were collected and assayed by TNF-α ELISA as detailed above.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196009&req=5

fig2: Analysis of TNF-α production by DCs in response to sHA stimulation. (A and B) Human monocyte–derived day 4 DCs were treated with 25 μg/ml sHA or 100 ng/ml LPS for the indicated times. After stimulation, (A) cells were harvested and RNA was prepared for TNF-α–specific RT-PCR, and (B) cell-free supernatants were collected and assayed by TNF-α ELISA. The dotted line shows the response to LPS, the solid line the response to sHA. Data represent the mean TNF-α release of triplicate values; pg/mg total protein ± SD. (C) DCs were incubated for 24 h with the indicated concentrations of sHA or 100 ng/ml LPS and cell-free supernatants were collected and assayed by TNF-α ELISA as detailed above.
Mentions: We have previously shown that DC stimulated with sHA secrete large quantities of proinflammatory cytokines, such as IL-1β, TNF-α, and IL-12, and that sHA-induced DC maturation is TNF-α dependent (5). To further characterize the time and dose dependence of sHA-induced TNF-α expression, we used RT-PCR to analyze TNF-α mRNA expression after sHA treatment of human DCs. Rapid induction of TNF-α mRNA was observed, which was detectable as early as 4 h after stimulation of DCs with either 25 μg/ml sHA or 100 ng/ml LPS (Fig. 2 A). At this dose the sHA-induced TNF-α expression was more long-lasting compared with that induced by LPS (Fig. 2 A). When the overall amount of TNF-α protein secreted into the culture medium after 24 h of stimulation with different concentrations of sHA was quantified by ELISA, there was a clear dose response in TNF-α production, with up to 4 ng/ml being produced in response to 50 μg/ml sHA (Fig. 2 B and C). The kinetics of TNF-α induction by sHA showed a late onset starting after 4–5 h of coincubation, reaching near-peak levels after 12 h, with a slight further increase up to 24 h. This effect was concentration dependent, as 10 μg/ml sHA sufficed to induce a significant TNF-α production, while saturation was observed at concentrations >20 μg/ml (Fig. 2 C). In contrast, TNF-α induction by 100 ng/ml LPS occurred much more rapidly, with significant amounts being produced after 30 min (Fig. 2 B). We conclude that the TNF-α response is a very sensitive parameter to measure the sHA-induced maturation of DC, therefore it was adopted as the major readout-system in further experiments.

Bottom Line: Western blot analysis revealed that sHA treatment resulted in distinct phosphorylation of p38/p42/44 MAP-kinases and nuclear translocation of nuclear factor (NF)-kappa B, all components of the TLR-4 signaling pathway.Finally, intravenous injection of sHA-induced DC emigration from the skin and their phenotypic and functional maturation in the spleen, again depending on the expression of TLR-4.In conclusion, this is the first report that polysaccharide degradation products of the extracellular matrix produced during inflammation might serve as an endogenous ligand for the TLR-4 complex on DCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, University of Freiburg, Freiburg D-79104, Germany. Termeer@haut.ukl.uni-freiburg.de

ABSTRACT
Low molecular weight fragmentation products of the polysaccharide of Hyaluronic acid (sHA) produced during inflammation have been shown to be potent activators of immunocompetent cells such as dendritic cells (DCs) and macrophages. Here we report that sHA induces maturation of DCs via the Toll-like receptor (TLR)-4, a receptor complex associated with innate immunity and host defense against bacterial infection. Bone marrow-derived DCs from C3H/HeJ and C57BL/10ScCr mice carrying mutant TLR-4 alleles were nonresponsive to sHA-induced phenotypic and functional maturation. Conversely, DCs from TLR-2-deficient mice were still susceptible to sHA. In accordance, addition of an anti-TLR-4 mAb to human monocyte-derived DCs blocked sHA-induced tumor necrosis factor alpha production. Western blot analysis revealed that sHA treatment resulted in distinct phosphorylation of p38/p42/44 MAP-kinases and nuclear translocation of nuclear factor (NF)-kappa B, all components of the TLR-4 signaling pathway. Blockade of this pathway by specific inhibitors completely abrogated the sHA-induced DC maturation. Finally, intravenous injection of sHA-induced DC emigration from the skin and their phenotypic and functional maturation in the spleen, again depending on the expression of TLR-4. In conclusion, this is the first report that polysaccharide degradation products of the extracellular matrix produced during inflammation might serve as an endogenous ligand for the TLR-4 complex on DCs.

Show MeSH
Related in: MedlinePlus