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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 induces phenotypic and functional maturation of human and mouse DCs. Human monocyte–derived day 4 DCs (A and B) or murine day 6 bone marrow–derived DCs (C and D) were used after 48-h incubation with 20 μg/ml sHA (bold line), 50 μg/ml HMW-HA (solid line), 100 ng/ml LPS (dotted line), or left untreated (broken line). (A and C) DCs were stained with mAbs directed against MHC class II (top) or B7–2 (CD86; lower) and analyzed by flow cytometry. A representative of five independent experiments is shown. (B and D) The cells were 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. *P > 0.001 compared with untreated DCs (−). A representative of four independent experiments is shown.
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fig1: sHA induces phenotypic and functional maturation of human and mouse DCs. Human monocyte–derived day 4 DCs (A and B) or murine day 6 bone marrow–derived DCs (C and D) were used after 48-h incubation with 20 μg/ml sHA (bold line), 50 μg/ml HMW-HA (solid line), 100 ng/ml LPS (dotted line), or left untreated (broken line). (A and C) DCs were stained with mAbs directed against MHC class II (top) or B7–2 (CD86; lower) and analyzed by flow cytometry. A representative of five independent experiments is shown. (B and D) The cells were 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. *P > 0.001 compared with untreated DCs (−). A representative of four independent experiments is shown.

Mentions: We and others have proposed that during inflammation high molecular weight hyaluronate is degraded by enzymes released from activated fibroblasts. These degradation products can deliver activation signals to DC and macrophages (2, 5). sHA fractions containing sugar units of 4–16 oligosaccharide size stimulated phenotypic and functional maturation of human monocyte-derived DCs, in contrast to HMW-HA (5). Maturation included upregulation of MHC class II and costimulatory B7-molecules, an enhanced allostimulatory capacity and an increased production of proinflammatory cytokines. This was independent of LPS-contamination, since preincubation with polymyxin B had no effect (5). Furthermore, LAL assays revealed that the maximal endotoxin content in our sHA preparations was <0.1 ng/ml, a concentration which is unable to induce functional or phenotypic DC maturation (reference 5, and data not shown). To facilitate further analysis of sHA-mediated DC maturation using distinct mouse strains, we set out to determine whether sHA also effectively activates murine DCs. Immature bone marrow–derived DCs from C57/BL-6 mice were coincubated for 48 h with either sHA, HMW-HA or LPS, or were left untreated. Only sHA, but not HMW-HA, induced maturation of murine DCs as shown by marked upregulation of MHC-class II (Iab) and costimulatory B7–2 molecules, resembling our observations in human DCs (Fig. 1 A and C). The maturation of sHA-stimulated DCs was accompanied by a dramatically enhanced capacity to stimulate the proliferation of naive alloreactive T cells in a standard MLR (Fig. 1 B and D). This phenotypic and functional maturation exactly matched the DC maturation induced by 100 ng/ml LPS (Fig. 1 D).


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 induces phenotypic and functional maturation of human and mouse DCs. Human monocyte–derived day 4 DCs (A and B) or murine day 6 bone marrow–derived DCs (C and D) were used after 48-h incubation with 20 μg/ml sHA (bold line), 50 μg/ml HMW-HA (solid line), 100 ng/ml LPS (dotted line), or left untreated (broken line). (A and C) DCs were stained with mAbs directed against MHC class II (top) or B7–2 (CD86; lower) and analyzed by flow cytometry. A representative of five independent experiments is shown. (B and D) The cells were 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. *P > 0.001 compared with untreated DCs (−). A representative of four independent experiments is shown.
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Related In: Results  -  Collection

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fig1: sHA induces phenotypic and functional maturation of human and mouse DCs. Human monocyte–derived day 4 DCs (A and B) or murine day 6 bone marrow–derived DCs (C and D) were used after 48-h incubation with 20 μg/ml sHA (bold line), 50 μg/ml HMW-HA (solid line), 100 ng/ml LPS (dotted line), or left untreated (broken line). (A and C) DCs were stained with mAbs directed against MHC class II (top) or B7–2 (CD86; lower) and analyzed by flow cytometry. A representative of five independent experiments is shown. (B and D) The cells were 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. *P > 0.001 compared with untreated DCs (−). A representative of four independent experiments is shown.
Mentions: We and others have proposed that during inflammation high molecular weight hyaluronate is degraded by enzymes released from activated fibroblasts. These degradation products can deliver activation signals to DC and macrophages (2, 5). sHA fractions containing sugar units of 4–16 oligosaccharide size stimulated phenotypic and functional maturation of human monocyte-derived DCs, in contrast to HMW-HA (5). Maturation included upregulation of MHC class II and costimulatory B7-molecules, an enhanced allostimulatory capacity and an increased production of proinflammatory cytokines. This was independent of LPS-contamination, since preincubation with polymyxin B had no effect (5). Furthermore, LAL assays revealed that the maximal endotoxin content in our sHA preparations was <0.1 ng/ml, a concentration which is unable to induce functional or phenotypic DC maturation (reference 5, and data not shown). To facilitate further analysis of sHA-mediated DC maturation using distinct mouse strains, we set out to determine whether sHA also effectively activates murine DCs. Immature bone marrow–derived DCs from C57/BL-6 mice were coincubated for 48 h with either sHA, HMW-HA or LPS, or were left untreated. Only sHA, but not HMW-HA, induced maturation of murine DCs as shown by marked upregulation of MHC-class II (Iab) and costimulatory B7–2 molecules, resembling our observations in human DCs (Fig. 1 A and C). The maturation of sHA-stimulated DCs was accompanied by a dramatically enhanced capacity to stimulate the proliferation of naive alloreactive T cells in a standard MLR (Fig. 1 B and D). This phenotypic and functional maturation exactly matched the DC maturation induced by 100 ng/ml LPS (Fig. 1 D).

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