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Cited by 1,378Immune Response and InflammationProteoglycans and glycosaminoglycans researchImmunotherapy and Immune Responses

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Oligosaccharides of Hyaluronan Activate Dendritic Cells via Toll-like Receptor 4

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Termeer, Christian, Benedix, Frauke, Sleeman, Jonathon, Fieber, Christina, Voith, Ursula, Ahrens, Thomas, Miyake, Kensuke, Freudenberg, Marina, Galanos, Christopher, Simon, Jan Christoph·2002·The Journal of Experimental Medicine
DOI 10.1084/jem.20001858PMC PMC2196009
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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 α 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)-κB, 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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Figures

Figure 1. 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.
Figure 1. 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.CC BY-NC-SA · PubMed Central
Figure 2. Analysis of TNF-α production by DCs in response to sHA stimulation. (A and B) Human monocyte–derived day 4 DCs were treated with 25 μg/ml sHA or 100 ng/ml LPS for the indicated times. After stimulation, (A) cells were harvested and RNA was prepared for TNF-α–specific RT-PCR, and (B) cell-free supernatants were collected and assayed by TNF-α ELISA. The dotted line shows the response to LPS, the solid line the response to sHA. Data represent the mean TNF-α release of triplicate values; pg/mg total protein ± SD. (C) DCs were incubated for 24 h with the indicated concentrations of sHA or 100 ng/ml LPS and cell-free supernatants were collected and assayed by TNF-α ELISA as detailed above.
Figure 2. Analysis of TNF-α production by DCs in response to sHA stimulation. (A and B) Human monocyte–derived day 4 DCs were treated with 25 μg/ml sHA or 100 ng/ml LPS for the indicated times. After stimulation, (A) cells were harvested and RNA was prepared for TNF-α–specific RT-PCR, and (B) cell-free supernatants were collected and assayed by TNF-α ELISA. The dotted line shows the response to LPS, the solid line the response to sHA. Data represent the mean TNF-α release of triplicate values; pg/mg total protein ± SD. (C) DCs were incubated for 24 h with the indicated concentrations of sHA or 100 ng/ml LPS and cell-free supernatants were collected and assayed by TNF-α ELISA as detailed above.CC BY-NC-SA · PubMed Central
Figure 3. sHA-mediated DC maturation requires p38-MAPK and P42/44-MAPK phosphorylation. (A and B) Human monocyte–derived day 4 DCs were treated with 50 μg/ml sHA, 100 μg/ml HMW-HA, or 100 ng/ml LPS for the times indicated. Western immunoblotting was performed using mAbs specific for the phosphorylated kinase (top) or total kinase content of the cells (bottom). Shown are blots for (A) p38 MAPK and (B) p42/44 MAPK. (C) Supernatants of DCs stimulated with 30 μg/ml sHA or 1 ng/ml LPS in the presence of the indicated concentrations of the MAPK inhibitors PD98059 (p38), SB 203580 (p42/44), Herbimycin A (src-like tyrosine kinases), or Wortmannin (PI3-kinase) were harvested after 7 h and screened for their TNF-α content by ELISA. Data represent the mean TNF-α release of triplicate wells; pg/mg total protein ± SD.
Figure 3. sHA-mediated DC maturation requires p38-MAPK and P42/44-MAPK phosphorylation. (A and B) Human monocyte–derived day 4 DCs were treated with 50 μg/ml sHA, 100 μg/ml HMW-HA, or 100 ng/ml LPS for the times indicated. Western immunoblotting was performed using mAbs specific for the phosphorylated kinase (top) or total kinase content of the cells (bottom). Shown are blots for (A) p38 MAPK and (B) p42/44 MAPK. (C) Supernatants of DCs stimulated with 30 μg/ml sHA or 1 ng/ml LPS in the presence of the indicated concentrations of the MAPK inhibitors PD98059 (p38), SB 203580 (p42/44), Herbimycin A (src-like tyrosine kinases), or Wortmannin (PI3-kinase) were harvested after 7 h and screened for their TNF-α content by ELISA. Data represent the mean TNF-α release of triplicate wells; pg/mg total protein ± SD.CC BY-NC-SA · PubMed Central
Figure 4. The sHA-induced DC maturation is NF-κB dependent. (A) Nuclear extracts were prepared from DCs treated with either HMW-HA (30 μg/ml), sHA (30 μg/ml), LPS (10 ng/ml), or left untreated. A total of 10 μg of protein from each nuclear extract was analyzed for NF-κB activity by EMSA. The arrow indicates specific NF-κB bands. (B) Supernatants of DCs stimulated with 30 μg/ml sHA (white bars) or 10 ng/ml LPS (black bars) in the presence of the indicated concentrations of the NF-κB inhibitor CAPE were harvested after 7 h and screened for their TNF-α content by ELISA. Data represent the mean TNF-α release of triplicate wells; pg/mg total protein ± SD.
Figure 4. The sHA-induced DC maturation is NF-κB dependent. (A) Nuclear extracts were prepared from DCs treated with either HMW-HA (30 μg/ml), sHA (30 μg/ml), LPS (10 ng/ml), or left untreated. A total of 10 μg of protein from each nuclear extract was analyzed for NF-κB activity by EMSA. The arrow indicates specific NF-κB bands. (B) Supernatants of DCs stimulated with 30 μg/ml sHA (white bars) or 10 ng/ml LPS (black bars) in the presence of the indicated concentrations of the NF-κB inhibitor CAPE were harvested after 7 h and screened for their TNF-α content by ELISA. Data represent the mean TNF-α release of triplicate wells; pg/mg total protein ± SD.CC BY-NC-SA · PubMed Central
Figure 5. 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.
Figure 5. 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.CC BY-NC-SA · PubMed Central
Figure 6. 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).
Figure 6. 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).CC BY-NC-SA · PubMed Central
Figure 7. 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.
Figure 7. 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.CC BY-NC-SA · PubMed Central
Figure 8. 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.
Figure 8. 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.CC BY-NC-SA · PubMed Central

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Metadata

Authors
Termeer, C., Benedix, F., Sleeman, J., Fieber, C., Voith, U., Ahrens, T., Miyake, K., Freudenberg, M., Galanos, C., & Simon, J. C.
Affiliations
  • University of Freiburg (DE)
  • 2Institute for Genetics, Forschungszentrum Karlsruhe, Karlsruhe D-76021, Germany
  • University of Basel (CH)
  • Institute of Medical Science, The University of Tokyo (JP)
  • Max Planck Society (DE)
  • 3Max-Planck Institute for Immunobiology, Freiburg D-79011, Germany
ORCID
Venue
The Journal of Experimental Medicine
Publisher
Rockefeller University Press
Published
2002-01-07
Volume
195
Issue
1
Pages
99-111
ISSN
  • 0022-1007 (ISSN-L)
  • 1540-9538
DOI
10.1084/jem.20001858
PMC
PMC2196009
OpenAlex
W2126659405
Semantic Scholar
44579905bd40557fb010b65248e3194bbd6a995d
Cited by
1,378
References
48
Citations by year
2026: 19 · 2025: 29 · 2024: 28 · 2023: 28 · 2022: 32 · 2021: 54 · 2020: 46 · 2019: 40 · 2018: 40 · 2017: 41 · 2016: 54 · 2015: 59 · 2014: 71 · 2013: 61 · 2012: 69
FWCI
18.95
Type
journal article
Language
en
Open access
Licence
cc-by-nc-sa
Locations
  • The Journal of Experimental Medicine (publishedVersion)
  • PubMed Central (publishedVersion, cc-by-nc-sa)
  • PubMed (publishedVersion)
  • Max Planck Digital Library (submittedVersion)
  • FreiDok plus (Universitätsbibliothek Freiburg) (submittedVersion)
  • MPG.PuRe (Max Planck Society) (submittedVersion)
Topics
  • Life Sciences › Immunology and Microbiology › Immunology › Immune Response and Inflammation
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Proteoglycans and glycosaminoglycans research
  • Life Sciences › Immunology and Microbiology › Immunology › Immunotherapy and Immune Responses
Keywords
hyaluronic acid, oligosaccharides, dendritic cell maturation, TLR4, TLR2, innate immunity, endogenous ligands, MAPK signaling, TNF-α, extracellular matrix degradation, inflammation
MeSH terms
Animals, Cell Differentiation, Dendritic Cells, Female, Humans, Hyaluronic Acid, Membrane Glycoproteins, Mice, Inbred C3H, Mice, Inbred C57BL, Oligosaccharides, Receptors, Cell Surface, Tumor Necrosis Factor-alpha, Signal Transduction, Down-Regulation, NF-kappa B, Mitogen-Activated Protein Kinases, Drosophila Proteins, Toll-Like Receptors, Toll-Like Receptor 2, Toll-Like Receptor 4, Mice

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