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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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In vivo relevance of the sHA-induced DC stimulation. (A) Full thickness skin was obtained from the outer epidermal sheet of the ears of C57BL/10ScSn wild-type (TLR+/+) and TLR-4−/− C57BL/10Cr mice at three animals per group. Ear sheets were floated on supplemented RPMI 1640 with or without addition of 30 μg/ml sHA or 100 ng/ml LPS. After 24 h, cells that had migrated into the culture medium were collected and double stained for CD11c and IaB expression. Large, highly CD11c and IaB-positive cells were considered as DCs and the percentage was calculated by flow cytometry. A representative of two independent experiments is shown. (B) 100 μg/animal sHA or 100 μl PBS was injected intravenous into the tail vein of C57BL/10ScSn wild-type (TLR+/+) and TLR-4 −/− C57BL/10Cr mice at three animals per group. The mice were killed after 12 h and large, highly CD11c-positive cells in the spleens were analyzed for their IaB and B7–2 expression by flow cytometry. Results are shown as mean fluorescence intensities (MFI) in the marked gates ± SD. Further, DCs were isolated from spleen cells and 5 × 103 DCs were restimulated for 4 d with 105 allogenic T cells from BALB/c mice. T cell proliferation was determined on day 4 by addition of 1 μCi of 3[H]thymidine for the final 18 h. Results are shown as counts per minute (CPM) ± SD of triplicate wells. A representative of two independent experiments is shown.
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fig8: In vivo relevance of the sHA-induced DC stimulation. (A) Full thickness skin was obtained from the outer epidermal sheet of the ears of C57BL/10ScSn wild-type (TLR+/+) and TLR-4−/− C57BL/10Cr mice at three animals per group. Ear sheets were floated on supplemented RPMI 1640 with or without addition of 30 μg/ml sHA or 100 ng/ml LPS. After 24 h, cells that had migrated into the culture medium were collected and double stained for CD11c and IaB expression. Large, highly CD11c and IaB-positive cells were considered as DCs and the percentage was calculated by flow cytometry. A representative of two independent experiments is shown. (B) 100 μg/animal sHA or 100 μl PBS was injected intravenous into the tail vein of C57BL/10ScSn wild-type (TLR+/+) and TLR-4 −/− C57BL/10Cr mice at three animals per group. The mice were killed after 12 h and large, highly CD11c-positive cells in the spleens were analyzed for their IaB and B7–2 expression by flow cytometry. Results are shown as mean fluorescence intensities (MFI) in the marked gates ± SD. Further, DCs were isolated from spleen cells and 5 × 103 DCs were restimulated for 4 d with 105 allogenic T cells from BALB/c mice. T cell proliferation was determined on day 4 by addition of 1 μCi of 3[H]thymidine for the final 18 h. Results are shown as counts per minute (CPM) ± SD of triplicate wells. A representative of two independent experiments is shown.

Mentions: The data presented so far suggesting that sHA-induced DC maturation is dependent on TLR-4 were produced using in vitro methods. Therefore, we set out to determine the in vivo relevance of sHA-induced DC maturation. In a first attempt, full thickness skin organ cultures were floated on medium containing 30 μg/ml sHA or 100 ng/ml LPS (Fig. 8 A). After 24 h, cells that had migrated into the culture medium were collected and double stained for CD11c and IaB expression. FACS® analysis of high forward scatter, highly CD11c/IaB-positive cells showed that a significant higher percentage of DCs appeared in the culture medium when sHA was added (Fig. 8 A). In contrast, ear skin from TLR-4–deficient mice did not show an enhanced DC emigration in response to sHA or LPS (Fig. 8 A). In a second set of experiments, 100 μg/animal sHA or 10 μg LPS were injected intravenously into C57BL/10ScCr and wild-type mice (Fig. 8 B). The mice were killed after 12 h and forward scatter high, highly CD11c-expressing cells were quantified in spleen cell suspensions for their IaB/B7–2 expression (Fig. 8 B). sHA induced an significant enrichment of highly Iab and B7–2 positive, activated DCs (Fig. 8 B). In contrast, injection of sHA into TLR-4–deficient mice did not result in an upregulation of Iab and B7–2 on splenic DCs (Fig. 8 B). To quantify their functional activity, DCs were purified from spleens and tested for their allostimulatory function using naive T cells from BALB/c mice as responders (Fig. 8 B). Thymidine incorporation clearly demonstrated an enhanced T cell proliferation induced by DCs isolated from spleens of wild-type mice that were injected with sHA compared with PBS treated controls. In contrast, DCs isolated from TLR-4–deficient C57BL/10ScCr injected with sHA did not induce an enhanced T cell proliferation (Fig. 8 B). From these results we conclude that exogenously applied sHA is able to induce DC activation in vivo and this activation is again mediated by TLR-4–dependent mechanisms.


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)

In vivo relevance of the sHA-induced DC stimulation. (A) Full thickness skin was obtained from the outer epidermal sheet of the ears of C57BL/10ScSn wild-type (TLR+/+) and TLR-4−/− C57BL/10Cr mice at three animals per group. Ear sheets were floated on supplemented RPMI 1640 with or without addition of 30 μg/ml sHA or 100 ng/ml LPS. After 24 h, cells that had migrated into the culture medium were collected and double stained for CD11c and IaB expression. Large, highly CD11c and IaB-positive cells were considered as DCs and the percentage was calculated by flow cytometry. A representative of two independent experiments is shown. (B) 100 μg/animal sHA or 100 μl PBS was injected intravenous into the tail vein of C57BL/10ScSn wild-type (TLR+/+) and TLR-4 −/− C57BL/10Cr mice at three animals per group. The mice were killed after 12 h and large, highly CD11c-positive cells in the spleens were analyzed for their IaB and B7–2 expression by flow cytometry. Results are shown as mean fluorescence intensities (MFI) in the marked gates ± SD. Further, DCs were isolated from spleen cells and 5 × 103 DCs were restimulated for 4 d with 105 allogenic T cells from BALB/c mice. T cell proliferation was determined on day 4 by addition of 1 μCi of 3[H]thymidine for the final 18 h. Results are shown as counts per minute (CPM) ± SD of triplicate wells. A representative of two independent experiments is shown.
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fig8: In vivo relevance of the sHA-induced DC stimulation. (A) Full thickness skin was obtained from the outer epidermal sheet of the ears of C57BL/10ScSn wild-type (TLR+/+) and TLR-4−/− C57BL/10Cr mice at three animals per group. Ear sheets were floated on supplemented RPMI 1640 with or without addition of 30 μg/ml sHA or 100 ng/ml LPS. After 24 h, cells that had migrated into the culture medium were collected and double stained for CD11c and IaB expression. Large, highly CD11c and IaB-positive cells were considered as DCs and the percentage was calculated by flow cytometry. A representative of two independent experiments is shown. (B) 100 μg/animal sHA or 100 μl PBS was injected intravenous into the tail vein of C57BL/10ScSn wild-type (TLR+/+) and TLR-4 −/− C57BL/10Cr mice at three animals per group. The mice were killed after 12 h and large, highly CD11c-positive cells in the spleens were analyzed for their IaB and B7–2 expression by flow cytometry. Results are shown as mean fluorescence intensities (MFI) in the marked gates ± SD. Further, DCs were isolated from spleen cells and 5 × 103 DCs were restimulated for 4 d with 105 allogenic T cells from BALB/c mice. T cell proliferation was determined on day 4 by addition of 1 μCi of 3[H]thymidine for the final 18 h. Results are shown as counts per minute (CPM) ± SD of triplicate wells. A representative of two independent experiments is shown.
Mentions: The data presented so far suggesting that sHA-induced DC maturation is dependent on TLR-4 were produced using in vitro methods. Therefore, we set out to determine the in vivo relevance of sHA-induced DC maturation. In a first attempt, full thickness skin organ cultures were floated on medium containing 30 μg/ml sHA or 100 ng/ml LPS (Fig. 8 A). After 24 h, cells that had migrated into the culture medium were collected and double stained for CD11c and IaB expression. FACS® analysis of high forward scatter, highly CD11c/IaB-positive cells showed that a significant higher percentage of DCs appeared in the culture medium when sHA was added (Fig. 8 A). In contrast, ear skin from TLR-4–deficient mice did not show an enhanced DC emigration in response to sHA or LPS (Fig. 8 A). In a second set of experiments, 100 μg/animal sHA or 10 μg LPS were injected intravenously into C57BL/10ScCr and wild-type mice (Fig. 8 B). The mice were killed after 12 h and forward scatter high, highly CD11c-expressing cells were quantified in spleen cell suspensions for their IaB/B7–2 expression (Fig. 8 B). sHA induced an significant enrichment of highly Iab and B7–2 positive, activated DCs (Fig. 8 B). In contrast, injection of sHA into TLR-4–deficient mice did not result in an upregulation of Iab and B7–2 on splenic DCs (Fig. 8 B). To quantify their functional activity, DCs were purified from spleens and tested for their allostimulatory function using naive T cells from BALB/c mice as responders (Fig. 8 B). Thymidine incorporation clearly demonstrated an enhanced T cell proliferation induced by DCs isolated from spleens of wild-type mice that were injected with sHA compared with PBS treated controls. In contrast, DCs isolated from TLR-4–deficient C57BL/10ScCr injected with sHA did not induce an enhanced T cell proliferation (Fig. 8 B). From these results we conclude that exogenously applied sHA is able to induce DC activation in vivo and this activation is again mediated by TLR-4–dependent mechanisms.

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