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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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sHA-mediated DC maturation requires p38-MAPK and P42/44-MAPK phosphorylation. (A and B) Human monocyte–derived day 4 DCs were treated with 50 μg/ml sHA, 100 μg/ml HMW-HA, or 100 ng/ml LPS for the times indicated. Western immunoblotting was performed using mAbs specific for the phosphorylated kinase (top) or total kinase content of the cells (bottom). Shown are blots for (A) p38 MAPK and (B) p42/44 MAPK. (C) Supernatants of DCs stimulated with 30 μg/ml sHA or 1 ng/ml LPS in the presence of the indicated concentrations of the MAPK inhibitors PD98059 (p38), SB 203580 (p42/44), Herbimycin A (src-like tyrosine kinases), or Wortmannin (PI3-kinase) 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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fig3: sHA-mediated DC maturation requires p38-MAPK and P42/44-MAPK phosphorylation. (A and B) Human monocyte–derived day 4 DCs were treated with 50 μg/ml sHA, 100 μg/ml HMW-HA, or 100 ng/ml LPS for the times indicated. Western immunoblotting was performed using mAbs specific for the phosphorylated kinase (top) or total kinase content of the cells (bottom). Shown are blots for (A) p38 MAPK and (B) p42/44 MAPK. (C) Supernatants of DCs stimulated with 30 μg/ml sHA or 1 ng/ml LPS in the presence of the indicated concentrations of the MAPK inhibitors PD98059 (p38), SB 203580 (p42/44), Herbimycin A (src-like tyrosine kinases), or Wortmannin (PI3-kinase) 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: It has been shown that DC maturation by LPS involves phosphorylation of p38 MAPK and p42/44 MAPK (13). To test whether activation of the MAPK pathway might be also involved in the sHA-induced DC maturation we analyzed p38 and p42/44 MAPK in DC lysates by Western blot analysis. After sHA treatment, but not HMW-HA treatment, p38 and p42/44 MAPK were rapidly phosphorylated within 1–2 h when compared with the total content of these MAPKs (Fig. 3 A and B). Then, we investigated whether p42/44 MAPK activation was required for the TNF-α production leading to DC maturation. DCs stimulated either with 30 μg/ml sHA or 100 ng/ml LPS were preincubated for 7 h with different concentrations of defined kinase inhibitors. Specifically, PD98059, which blocks p42/44 MAPKs, SB-203580, a selective inhibitor of p38-MAPKs, Herbimycin A, an inhibitor of src-like protein tyrosine kinases, and Wortmannin, a phosphatidylinositol (PI)3-kinase inhibitor were used as described previously (28–31). ELISA assays showed a dose-dependent decrease in TNF-α production after preincubation with each of the different MAPK inhibitors, whereas Wortmannin had no effect, suggesting that p38 and p42/44 MAPKs, but not PI3-kinase are involved in the signaling pathway induced by sHA (Fig. 3 C). Since activation of PI3-kinase is involved in the control of intracellular calcium stores, the latter finding is consistent with our results that sHA treatment of DCs does not induce intracellular calcium fluxes as analyzed by Fura labeling (data not shown).


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)

sHA-mediated DC maturation requires p38-MAPK and P42/44-MAPK phosphorylation. (A and B) Human monocyte–derived day 4 DCs were treated with 50 μg/ml sHA, 100 μg/ml HMW-HA, or 100 ng/ml LPS for the times indicated. Western immunoblotting was performed using mAbs specific for the phosphorylated kinase (top) or total kinase content of the cells (bottom). Shown are blots for (A) p38 MAPK and (B) p42/44 MAPK. (C) Supernatants of DCs stimulated with 30 μg/ml sHA or 1 ng/ml LPS in the presence of the indicated concentrations of the MAPK inhibitors PD98059 (p38), SB 203580 (p42/44), Herbimycin A (src-like tyrosine kinases), or Wortmannin (PI3-kinase) 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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fig3: sHA-mediated DC maturation requires p38-MAPK and P42/44-MAPK phosphorylation. (A and B) Human monocyte–derived day 4 DCs were treated with 50 μg/ml sHA, 100 μg/ml HMW-HA, or 100 ng/ml LPS for the times indicated. Western immunoblotting was performed using mAbs specific for the phosphorylated kinase (top) or total kinase content of the cells (bottom). Shown are blots for (A) p38 MAPK and (B) p42/44 MAPK. (C) Supernatants of DCs stimulated with 30 μg/ml sHA or 1 ng/ml LPS in the presence of the indicated concentrations of the MAPK inhibitors PD98059 (p38), SB 203580 (p42/44), Herbimycin A (src-like tyrosine kinases), or Wortmannin (PI3-kinase) 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: It has been shown that DC maturation by LPS involves phosphorylation of p38 MAPK and p42/44 MAPK (13). To test whether activation of the MAPK pathway might be also involved in the sHA-induced DC maturation we analyzed p38 and p42/44 MAPK in DC lysates by Western blot analysis. After sHA treatment, but not HMW-HA treatment, p38 and p42/44 MAPK were rapidly phosphorylated within 1–2 h when compared with the total content of these MAPKs (Fig. 3 A and B). Then, we investigated whether p42/44 MAPK activation was required for the TNF-α production leading to DC maturation. DCs stimulated either with 30 μg/ml sHA or 100 ng/ml LPS were preincubated for 7 h with different concentrations of defined kinase inhibitors. Specifically, PD98059, which blocks p42/44 MAPKs, SB-203580, a selective inhibitor of p38-MAPKs, Herbimycin A, an inhibitor of src-like protein tyrosine kinases, and Wortmannin, a phosphatidylinositol (PI)3-kinase inhibitor were used as described previously (28–31). ELISA assays showed a dose-dependent decrease in TNF-α production after preincubation with each of the different MAPK inhibitors, whereas Wortmannin had no effect, suggesting that p38 and p42/44 MAPKs, but not PI3-kinase are involved in the signaling pathway induced by sHA (Fig. 3 C). Since activation of PI3-kinase is involved in the control of intracellular calcium stores, the latter finding is consistent with our results that sHA treatment of DCs does not induce intracellular calcium fluxes as analyzed by Fura labeling (data not shown).

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