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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 stimulation is not dependent on TLR-2 expression. Day 6 bone marrow–derived DCs from C57BL/6 or C57BL/6TLR-2−/− were incubated for 48 h with the indicated concentrations of highly purified LPS from Salmonella abortus equi or sHA. (A) The cell free supernatants were screened for their TNF-α content by ELISA. Data represent the mean TNF-α release of triplicate wells; pg/mg total protein ± SD. (B) DCs were treated for 48 h with the same substances described in A, washed, and coincubated for 4 d with 105 alloreactive T cells at a DC/TC ratio of 1:20. T cell proliferation was determined on day 5 by addition of 1 μCi of 3[H]thymidine for the final 18 h. Results are shown in counts per minute (CPM) ± SD of triplicate wells.
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fig7: sHA-mediated DC stimulation is not dependent on TLR-2 expression. Day 6 bone marrow–derived DCs from C57BL/6 or C57BL/6TLR-2−/− were incubated for 48 h with the indicated concentrations of highly purified LPS from Salmonella abortus equi or sHA. (A) The cell free supernatants were screened for their TNF-α content by ELISA. Data represent the mean TNF-α release of triplicate wells; pg/mg total protein ± SD. (B) DCs were treated for 48 h with the same substances described in A, washed, and coincubated for 4 d with 105 alloreactive T cells at a DC/TC ratio of 1:20. T cell proliferation was determined on day 5 by addition of 1 μCi of 3[H]thymidine for the final 18 h. Results are shown in counts per minute (CPM) ± SD of triplicate wells.

Mentions: TLR-2 is a receptor that has been associated with macrophage activation by membrane lipoproteins/lipopeptides from Borrelia burgdorferi, Treponema pallidum, and Mycoplasma fermentans (14, 33). To examine whether TLR-2 plays a role in sHA-induced DC maturation, DCs from TLR-2–deficient (14) and wild-type mice were generated from bone marrow. These cells were treated with either LPS or sHA and their ability to secrete TNF-α in response to these substances was compared. TLR-2–deficient DCs responded well to LPS and sHA (Fig. 7 A). These data were confirmed at the functional level, since in a standard MLR, the allostimulatory capacity of the DCs from mutant mice was significantly enhanced upon sHA and LPS treatment (Fig. 7 B). Taken together, these data suggest that sHA-mediated DC stimulation is dependent on TLR-4 but not on TLR-2, since the presence of a nonfunctional mutated TLR-4 receptor in DCs could not support sHA-induced DC maturation, while TLR-2–deficient DCs responded to sHA in a similar manner to wild-type DCs.


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 stimulation is not dependent on TLR-2 expression. Day 6 bone marrow–derived DCs from C57BL/6 or C57BL/6TLR-2−/− were incubated for 48 h with the indicated concentrations of highly purified LPS from Salmonella abortus equi or sHA. (A) The cell free supernatants were screened for their TNF-α content by ELISA. Data represent the mean TNF-α release of triplicate wells; pg/mg total protein ± SD. (B) DCs were treated for 48 h with the same substances described in A, washed, and coincubated for 4 d with 105 alloreactive T cells at a DC/TC ratio of 1:20. T cell proliferation was determined on day 5 by addition of 1 μCi of 3[H]thymidine for the final 18 h. Results are shown in counts per minute (CPM) ± SD of triplicate wells.
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Related In: Results  -  Collection

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fig7: sHA-mediated DC stimulation is not dependent on TLR-2 expression. Day 6 bone marrow–derived DCs from C57BL/6 or C57BL/6TLR-2−/− were incubated for 48 h with the indicated concentrations of highly purified LPS from Salmonella abortus equi or sHA. (A) The cell free supernatants were screened for their TNF-α content by ELISA. Data represent the mean TNF-α release of triplicate wells; pg/mg total protein ± SD. (B) DCs were treated for 48 h with the same substances described in A, washed, and coincubated for 4 d with 105 alloreactive T cells at a DC/TC ratio of 1:20. T cell proliferation was determined on day 5 by addition of 1 μCi of 3[H]thymidine for the final 18 h. Results are shown in counts per minute (CPM) ± SD of triplicate wells.
Mentions: TLR-2 is a receptor that has been associated with macrophage activation by membrane lipoproteins/lipopeptides from Borrelia burgdorferi, Treponema pallidum, and Mycoplasma fermentans (14, 33). To examine whether TLR-2 plays a role in sHA-induced DC maturation, DCs from TLR-2–deficient (14) and wild-type mice were generated from bone marrow. These cells were treated with either LPS or sHA and their ability to secrete TNF-α in response to these substances was compared. TLR-2–deficient DCs responded well to LPS and sHA (Fig. 7 A). These data were confirmed at the functional level, since in a standard MLR, the allostimulatory capacity of the DCs from mutant mice was significantly enhanced upon sHA and LPS treatment (Fig. 7 B). Taken together, these data suggest that sHA-mediated DC stimulation is dependent on TLR-4 but not on TLR-2, since the presence of a nonfunctional mutated TLR-4 receptor in DCs could not support sHA-induced DC maturation, while TLR-2–deficient DCs responded to sHA in a similar manner to wild-type DCs.

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