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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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Related in: MedlinePlus

The sHA-induced DC maturation is NF-κB dependent. (A) Nuclear extracts were prepared from DCs treated with either HMW-HA (30 μg/ml), sHA (30 μg/ml), LPS (10 ng/ml), or left untreated. A total of 10 μg of protein from each nuclear extract was analyzed for NF-κB activity by EMSA. The arrow indicates specific NF-κB bands. (B) Supernatants of DCs stimulated with 30 μg/ml sHA (white bars) or 10 ng/ml LPS (black bars) in the presence of the indicated concentrations of the NF-κB inhibitor CAPE were harvested after 7 h and screened for their TNF-α content by ELISA. Data represent the mean TNF-α release of triplicate wells; pg/mg total protein ± SD.
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fig4: The sHA-induced DC maturation is NF-κB dependent. (A) Nuclear extracts were prepared from DCs treated with either HMW-HA (30 μg/ml), sHA (30 μg/ml), LPS (10 ng/ml), or left untreated. A total of 10 μg of protein from each nuclear extract was analyzed for NF-κB activity by EMSA. The arrow indicates specific NF-κB bands. (B) Supernatants of DCs stimulated with 30 μg/ml sHA (white bars) or 10 ng/ml LPS (black bars) in the presence of the indicated concentrations of the NF-κB inhibitor CAPE were harvested after 7 h and screened for their TNF-α content by ELISA. Data represent the mean TNF-α release of triplicate wells; pg/mg total protein ± SD.

Mentions: To determine whether the sHA-induced DC maturation involves NF-κB translocation to the nucleus, EMSA of nuclear extracts were performed. As shown in Fig. 4 A, sHA induced NF-κB activation in a time-dependent manner, with a significant signal obtained as early as 30 min after sHA treatment. Furthermore, coincubation with the specific NF-κB inhibitor CAPE dose dependently inhibited the ability of DCs to produce TNF-α and their upregulation of HLA-DR (Fig. 4 B and data not shown), with an almost total inhibitory effect observed at a concentration of 20 μg/ml. As expected, NF-κB liberation from the cytosol was enhanced by LPS, with strong NF-κB signaling being observed after 30 min of incubation with LPS (Fig. 4 A). Taken together, these data suggest that phosphorylation of p38 and p42/44 MAPK as well as nuclear translocation of NF-κB are prerequisites for the sHA-induced TNF-α production by DC.


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)

The sHA-induced DC maturation is NF-κB dependent. (A) Nuclear extracts were prepared from DCs treated with either HMW-HA (30 μg/ml), sHA (30 μg/ml), LPS (10 ng/ml), or left untreated. A total of 10 μg of protein from each nuclear extract was analyzed for NF-κB activity by EMSA. The arrow indicates specific NF-κB bands. (B) Supernatants of DCs stimulated with 30 μg/ml sHA (white bars) or 10 ng/ml LPS (black bars) in the presence of the indicated concentrations of the NF-κB inhibitor CAPE were harvested after 7 h and screened for their TNF-α content by ELISA. Data represent the mean TNF-α release of triplicate wells; pg/mg total protein ± SD.
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Related In: Results  -  Collection

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fig4: The sHA-induced DC maturation is NF-κB dependent. (A) Nuclear extracts were prepared from DCs treated with either HMW-HA (30 μg/ml), sHA (30 μg/ml), LPS (10 ng/ml), or left untreated. A total of 10 μg of protein from each nuclear extract was analyzed for NF-κB activity by EMSA. The arrow indicates specific NF-κB bands. (B) Supernatants of DCs stimulated with 30 μg/ml sHA (white bars) or 10 ng/ml LPS (black bars) in the presence of the indicated concentrations of the NF-κB inhibitor CAPE were harvested after 7 h and screened for their TNF-α content by ELISA. Data represent the mean TNF-α release of triplicate wells; pg/mg total protein ± SD.
Mentions: To determine whether the sHA-induced DC maturation involves NF-κB translocation to the nucleus, EMSA of nuclear extracts were performed. As shown in Fig. 4 A, sHA induced NF-κB activation in a time-dependent manner, with a significant signal obtained as early as 30 min after sHA treatment. Furthermore, coincubation with the specific NF-κB inhibitor CAPE dose dependently inhibited the ability of DCs to produce TNF-α and their upregulation of HLA-DR (Fig. 4 B and data not shown), with an almost total inhibitory effect observed at a concentration of 20 μg/ml. As expected, NF-κB liberation from the cytosol was enhanced by LPS, with strong NF-κB signaling being observed after 30 min of incubation with LPS (Fig. 4 A). Taken together, these data suggest that phosphorylation of p38 and p42/44 MAPK as well as nuclear translocation of NF-κB are prerequisites for the sHA-induced TNF-α production by DC.

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