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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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DCs derived from TLR-4–deficient mice are not sensitive to sHA stimulation. (A) Bone marrow–derived DCs were obtained from either C3H/HeN (wild-type) and C3H/HeJ (TLR-4 mutant) or C57BL/10ScSn (wild-type) and C57BL/10ScCr (TLR-4 mutant). On day 6 of culture the DCs were either incubated with 100 ng/ml of bacterial cell wall lysates containing LPS and other substances such as lipoprotein, or 100 ng/ml highly purified LPS from Salmonella abortus equi or Salmonella minessota strain R595 as positive and negative controls. Alternatively, 10 or 50 μg/ml sHA or 100 μg/ml HMW-HA were added. After 48 h of treatment 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. (C) Day 6 bone marrow–derived DCs of either C57BL/10Sn (black bars) or C57BL/10Cr (white bars) were incubated for 24 h with 30 μg/ml sHA or 100 ng/ml LPS and/or pretreated with 10 mg/ml polymyxin B (polyB) 1 h before stimulation. After 24 h of incubation DCs were stained for Iab, B7–1, and B7–2 expression and analyzed by flow cytometry. Results are shown a mean fluorescence intensities (MFI).
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fig6: DCs derived from TLR-4–deficient mice are not sensitive to sHA stimulation. (A) Bone marrow–derived DCs were obtained from either C3H/HeN (wild-type) and C3H/HeJ (TLR-4 mutant) or C57BL/10ScSn (wild-type) and C57BL/10ScCr (TLR-4 mutant). On day 6 of culture the DCs were either incubated with 100 ng/ml of bacterial cell wall lysates containing LPS and other substances such as lipoprotein, or 100 ng/ml highly purified LPS from Salmonella abortus equi or Salmonella minessota strain R595 as positive and negative controls. Alternatively, 10 or 50 μg/ml sHA or 100 μg/ml HMW-HA were added. After 48 h of treatment 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. (C) Day 6 bone marrow–derived DCs of either C57BL/10Sn (black bars) or C57BL/10Cr (white bars) were incubated for 24 h with 30 μg/ml sHA or 100 ng/ml LPS and/or pretreated with 10 mg/ml polymyxin B (polyB) 1 h before stimulation. After 24 h of incubation DCs were stained for Iab, B7–1, and B7–2 expression and analyzed by flow cytometry. Results are shown a mean fluorescence intensities (MFI).

Mentions: The mouse strains C3H/HeJ and C57BL10/ScCr are nonresponsive to LPS due to a mutation or lack of the TLR-4 receptor, respectively (19). We used these mice to further characterize the function of TLR-4 during sHA-mediated DC stimulation. Bone marrow–derived DCs from C3H/HeJ, C57BL/10ScCr, and wild-type mice were prepared and immunophenotypically analyzed. We found no differences in expression levels of MHC class II, costimulatory molecules B7–1/B7–2 or CD11c in DCs from TLR-4–deficient or wild-type mice (data not shown). Moreover, the DCs from the mutant mice were functionally active, since they produced significant amounts of TNF-α after coincubation with bacterial crude cell wall extracts which were shown to activate macrophages from TLR-4–deficient mice (17), although this amount was lower than that produced by wild-type DCs (Fig. 6 A). However, while sHA dose dependently stimulated TNF-α production in wild-type DCs as expected, DCs from mutant mice were completely resistant to sHA stimulation as well as to purified LPS from S. abortus equi or S. minnesota R595 (Fig. 6 A). We next examined whether the nonresponsiveness of DCs from TLR-4 mutant mice to sHA-stimulation is functionally relevant to their T cell stimulatory function. In a standard MLR, the allostimulatory capacity of the DCs from mutant mice was significantly lowered on sHA treatment when compared with wild-type DCs (Fig. 6 B). In comparison, HMW-HA had no effect on either the TNF-α release or the T cell stimulatory capacity of both wild-type and mutant DCs, even at concentrations of 100 μg/ml (Fig. 6). Since TLR-4 is the major receptor for LPS and concentrations as low as 50 pg/ml have been described to activate cells of the myelomonocytic lineage (32), we wished to exclude the possibility of endotoxin contamination in the sHA preparations used in this study. LAL assays showed a maximum endotoxin content of 0.1 ng/ml in our 1 mg/ml sHA stock solutions, resulting in a maximum possible contamination throughout the assays of 0.005 ng/ml in the 50 μg/ml concentration of sHA. Titration experiments with purified LPS from S. abortus equi were performed, showing that DCs under the culture conditions used in our experiments did not respond to LPS concentrations <0.2 ng/ml (data not shown). Furthermore, blocking experiments were performed by preincubating the DCs with 10 μg/ml of the LPS-inhibitor polymyxin B before sHA stimulation (Fig. 6 C). Polymyxin B had no effect on the sHA-induced upregulation of IAb, B7–1, or B7–2, but in the same experiment inhibited the LPS effect almost completely (Fig. 6 C).


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)

DCs derived from TLR-4–deficient mice are not sensitive to sHA stimulation. (A) Bone marrow–derived DCs were obtained from either C3H/HeN (wild-type) and C3H/HeJ (TLR-4 mutant) or C57BL/10ScSn (wild-type) and C57BL/10ScCr (TLR-4 mutant). On day 6 of culture the DCs were either incubated with 100 ng/ml of bacterial cell wall lysates containing LPS and other substances such as lipoprotein, or 100 ng/ml highly purified LPS from Salmonella abortus equi or Salmonella minessota strain R595 as positive and negative controls. Alternatively, 10 or 50 μg/ml sHA or 100 μg/ml HMW-HA were added. After 48 h of treatment 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. (C) Day 6 bone marrow–derived DCs of either C57BL/10Sn (black bars) or C57BL/10Cr (white bars) were incubated for 24 h with 30 μg/ml sHA or 100 ng/ml LPS and/or pretreated with 10 mg/ml polymyxin B (polyB) 1 h before stimulation. After 24 h of incubation DCs were stained for Iab, B7–1, and B7–2 expression and analyzed by flow cytometry. Results are shown a mean fluorescence intensities (MFI).
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fig6: DCs derived from TLR-4–deficient mice are not sensitive to sHA stimulation. (A) Bone marrow–derived DCs were obtained from either C3H/HeN (wild-type) and C3H/HeJ (TLR-4 mutant) or C57BL/10ScSn (wild-type) and C57BL/10ScCr (TLR-4 mutant). On day 6 of culture the DCs were either incubated with 100 ng/ml of bacterial cell wall lysates containing LPS and other substances such as lipoprotein, or 100 ng/ml highly purified LPS from Salmonella abortus equi or Salmonella minessota strain R595 as positive and negative controls. Alternatively, 10 or 50 μg/ml sHA or 100 μg/ml HMW-HA were added. After 48 h of treatment 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. (C) Day 6 bone marrow–derived DCs of either C57BL/10Sn (black bars) or C57BL/10Cr (white bars) were incubated for 24 h with 30 μg/ml sHA or 100 ng/ml LPS and/or pretreated with 10 mg/ml polymyxin B (polyB) 1 h before stimulation. After 24 h of incubation DCs were stained for Iab, B7–1, and B7–2 expression and analyzed by flow cytometry. Results are shown a mean fluorescence intensities (MFI).
Mentions: The mouse strains C3H/HeJ and C57BL10/ScCr are nonresponsive to LPS due to a mutation or lack of the TLR-4 receptor, respectively (19). We used these mice to further characterize the function of TLR-4 during sHA-mediated DC stimulation. Bone marrow–derived DCs from C3H/HeJ, C57BL/10ScCr, and wild-type mice were prepared and immunophenotypically analyzed. We found no differences in expression levels of MHC class II, costimulatory molecules B7–1/B7–2 or CD11c in DCs from TLR-4–deficient or wild-type mice (data not shown). Moreover, the DCs from the mutant mice were functionally active, since they produced significant amounts of TNF-α after coincubation with bacterial crude cell wall extracts which were shown to activate macrophages from TLR-4–deficient mice (17), although this amount was lower than that produced by wild-type DCs (Fig. 6 A). However, while sHA dose dependently stimulated TNF-α production in wild-type DCs as expected, DCs from mutant mice were completely resistant to sHA stimulation as well as to purified LPS from S. abortus equi or S. minnesota R595 (Fig. 6 A). We next examined whether the nonresponsiveness of DCs from TLR-4 mutant mice to sHA-stimulation is functionally relevant to their T cell stimulatory function. In a standard MLR, the allostimulatory capacity of the DCs from mutant mice was significantly lowered on sHA treatment when compared with wild-type DCs (Fig. 6 B). In comparison, HMW-HA had no effect on either the TNF-α release or the T cell stimulatory capacity of both wild-type and mutant DCs, even at concentrations of 100 μg/ml (Fig. 6). Since TLR-4 is the major receptor for LPS and concentrations as low as 50 pg/ml have been described to activate cells of the myelomonocytic lineage (32), we wished to exclude the possibility of endotoxin contamination in the sHA preparations used in this study. LAL assays showed a maximum endotoxin content of 0.1 ng/ml in our 1 mg/ml sHA stock solutions, resulting in a maximum possible contamination throughout the assays of 0.005 ng/ml in the 50 μg/ml concentration of sHA. Titration experiments with purified LPS from S. abortus equi were performed, showing that DCs under the culture conditions used in our experiments did not respond to LPS concentrations <0.2 ng/ml (data not shown). Furthermore, blocking experiments were performed by preincubating the DCs with 10 μg/ml of the LPS-inhibitor polymyxin B before sHA stimulation (Fig. 6 C). Polymyxin B had no effect on the sHA-induced upregulation of IAb, B7–1, or B7–2, but in the same experiment inhibited the LPS effect almost completely (Fig. 6 C).

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