Limits...
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.

Show MeSH

Related in: MedlinePlus

TLR-4 mediates TNF-α production of sHA-stimulated human DCs and is downregulated after activation. (A) Anti–TLR-4 antibodies inhibit TNF-α production by DCs in response to sHA (left) and LPS (right). DCs were either preincubated for 30 or 60 min with 10 or 20 μg/ml of the anti–TLR-4 mAb HTA 125 as indicated, a matched IgG-isotype control (black circles) or left untreated (white circles). The cells were then stimulated with 30 μg/ml sHA (left) or 100 ng/ml LPS (right) for the times (h) indicated. Supernatants were harvested and screened for their TNF-α content by ELISA as described previously. Data represent the mean TNF-α release of triplicate wells; ng/mg total protein. (B) Total mRNA from human DCs treated either with 100 ng/ml LPS or 30 μg/ml sHA for the indicated times was analyzed by RT-PCR. The top shows TLR-2, the middle TLR-4 regulation, the bottom shows β-actin control. (C) DCs were analyzed by FACS® for their surface-expression of TLR-4 on: untreated control DCs (solid line), HMW-HA–treated DCs (30 μg/ml; dashed line), sHA-treated DCs (30 μg/ml; bold line), and LPS-treated DC (1 ng/ml; dotted line). Dot-dash line: DCs stained with secondary antibody alone. A representative of three independent experiments is shown.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196009&req=5

fig5: TLR-4 mediates TNF-α production of sHA-stimulated human DCs and is downregulated after activation. (A) Anti–TLR-4 antibodies inhibit TNF-α production by DCs in response to sHA (left) and LPS (right). DCs were either preincubated for 30 or 60 min with 10 or 20 μg/ml of the anti–TLR-4 mAb HTA 125 as indicated, a matched IgG-isotype control (black circles) or left untreated (white circles). The cells were then stimulated with 30 μg/ml sHA (left) or 100 ng/ml LPS (right) for the times (h) indicated. Supernatants were harvested and screened for their TNF-α content by ELISA as described previously. Data represent the mean TNF-α release of triplicate wells; ng/mg total protein. (B) Total mRNA from human DCs treated either with 100 ng/ml LPS or 30 μg/ml sHA for the indicated times was analyzed by RT-PCR. The top shows TLR-2, the middle TLR-4 regulation, the bottom shows β-actin control. (C) DCs were analyzed by FACS® for their surface-expression of TLR-4 on: untreated control DCs (solid line), HMW-HA–treated DCs (30 μg/ml; dashed line), sHA-treated DCs (30 μg/ml; bold line), and LPS-treated DC (1 ng/ml; dotted line). Dot-dash line: DCs stained with secondary antibody alone. A representative of three independent experiments is shown.

Mentions: The TLR-4 family, in particular the TLR-4 complex, has recently been described as being involved in the LPS-dependent activation of DCs during bacterial infection (13). We investigated whether TLR-4 might also be involved in sHA-induced activation of DCs. The anti–TLR-4 mAb HTA125 is able to block TLR-4 ligation (23). Therefore, we analyzed what effect this antibody has on TNF-α production by DCs in response to sHA. Preincubation of human DC for 30 min with 10 μg/ml HTA125 mAb, a concentration previously shown to effectively block TLR-4 ligation (23), was able to reduce the sHA-induced TNFα release by these cells for up to 7 h, as found for LPS (Fig. 5 A). A higher concentration of HTA 125 (20 μg/ml) or a longer preincubation time of 60 min could enhance the blocking effect to some extent (Fig. 5 A). These results provide first evidence that TLR-4 is involved in the intracellular signaling and maturation of DCs induced by sHA.


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)

TLR-4 mediates TNF-α production of sHA-stimulated human DCs and is downregulated after activation. (A) Anti–TLR-4 antibodies inhibit TNF-α production by DCs in response to sHA (left) and LPS (right). DCs were either preincubated for 30 or 60 min with 10 or 20 μg/ml of the anti–TLR-4 mAb HTA 125 as indicated, a matched IgG-isotype control (black circles) or left untreated (white circles). The cells were then stimulated with 30 μg/ml sHA (left) or 100 ng/ml LPS (right) for the times (h) indicated. Supernatants were harvested and screened for their TNF-α content by ELISA as described previously. Data represent the mean TNF-α release of triplicate wells; ng/mg total protein. (B) Total mRNA from human DCs treated either with 100 ng/ml LPS or 30 μg/ml sHA for the indicated times was analyzed by RT-PCR. The top shows TLR-2, the middle TLR-4 regulation, the bottom shows β-actin control. (C) DCs were analyzed by FACS® for their surface-expression of TLR-4 on: untreated control DCs (solid line), HMW-HA–treated DCs (30 μg/ml; dashed line), sHA-treated DCs (30 μg/ml; bold line), and LPS-treated DC (1 ng/ml; dotted line). Dot-dash line: DCs stained with secondary antibody alone. A representative of three independent experiments is shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196009&req=5

fig5: TLR-4 mediates TNF-α production of sHA-stimulated human DCs and is downregulated after activation. (A) Anti–TLR-4 antibodies inhibit TNF-α production by DCs in response to sHA (left) and LPS (right). DCs were either preincubated for 30 or 60 min with 10 or 20 μg/ml of the anti–TLR-4 mAb HTA 125 as indicated, a matched IgG-isotype control (black circles) or left untreated (white circles). The cells were then stimulated with 30 μg/ml sHA (left) or 100 ng/ml LPS (right) for the times (h) indicated. Supernatants were harvested and screened for their TNF-α content by ELISA as described previously. Data represent the mean TNF-α release of triplicate wells; ng/mg total protein. (B) Total mRNA from human DCs treated either with 100 ng/ml LPS or 30 μg/ml sHA for the indicated times was analyzed by RT-PCR. The top shows TLR-2, the middle TLR-4 regulation, the bottom shows β-actin control. (C) DCs were analyzed by FACS® for their surface-expression of TLR-4 on: untreated control DCs (solid line), HMW-HA–treated DCs (30 μg/ml; dashed line), sHA-treated DCs (30 μg/ml; bold line), and LPS-treated DC (1 ng/ml; dotted line). Dot-dash line: DCs stained with secondary antibody alone. A representative of three independent experiments is shown.
Mentions: The TLR-4 family, in particular the TLR-4 complex, has recently been described as being involved in the LPS-dependent activation of DCs during bacterial infection (13). We investigated whether TLR-4 might also be involved in sHA-induced activation of DCs. The anti–TLR-4 mAb HTA125 is able to block TLR-4 ligation (23). Therefore, we analyzed what effect this antibody has on TNF-α production by DCs in response to sHA. Preincubation of human DC for 30 min with 10 μg/ml HTA125 mAb, a concentration previously shown to effectively block TLR-4 ligation (23), was able to reduce the sHA-induced TNFα release by these cells for up to 7 h, as found for LPS (Fig. 5 A). A higher concentration of HTA 125 (20 μg/ml) or a longer preincubation time of 60 min could enhance the blocking effect to some extent (Fig. 5 A). These results provide first evidence that TLR-4 is involved in the intracellular signaling and maturation of DCs induced by sHA.

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