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Cited by 723Advanced Glycation End Products researchImmune Response and InflammationNeutrophil, Myeloperoxidase and Oxidative Mechanisms

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Mutually exclusive redox forms of HMGB1 promote cell recruitment or proinflammatory cytokine release

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Venereau, Emilie, Casalgrandi, Maura, Schiraldi, Milena, Antoine, Daniel J., Cattaneo, Angela, De Marchis, Francesco, Liu, Jaron, Antonelli, Antonella, Preti, Alessandro, Raeli, Lorenzo, Shams, Sara Samadi, Yang, Huan, Varani, Luca, Andersson, Ulf, Tracey, Kevin J., Bachi, Angela, Uguccioni, Mariagrazia, Bianchi, Marco E.·2012·Journal of Experimental Medicine
DOI 10.1084/jem.20120189PMC PMC3428943
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Abstract

Tissue damage causes inflammation, by recruiting leukocytes and activating them to release proinflammatory mediators. We show that high-mobility group box 1 protein (HMGB1) orchestrates both processes by switching among mutually exclusive redox states. Reduced cysteines make HMGB1 a chemoattractant, whereas a disulfide bond makes it a proinflammatory cytokine and further cysteine oxidation to sulfonates by reactive oxygen species abrogates both activities. We show that leukocyte recruitment and activation can be separated. A nonoxidizable HMGB1 mutant in which serines replace all cysteines (3S-HMGB1) does not promote cytokine production, but is more effective than wild-type HMGB1 in recruiting leukocytes in vivo. BoxA, a HMGB1 inhibitor, interferes with leukocyte recruitment but not with activation. We detected the different redox forms of HMGB1 ex vivo within injured muscle. HMGB1 is completely reduced at first and disulfide-bonded later. Thus, HMGB1 orchestrates both key events in sterile inflammation, leukocyte recruitment and their induction to secrete inflammatory cytokines, by adopting mutually exclusive redox states.

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Figures

Figure 1. Characterization of HMGB1 redox states. (A) Mass spectrometric characterization of HMGB1 purified with DTT (top) or without DTT (bottom). Characterization of C23: MS-MS trace of the peptide containing amino acids 13–24, with an iodoacetamide adduct indicating reduced C23 (top) or with a NEM adduct after DTT reduction of a disulfide bond (bottom). Characterization of C45: MS-MS trace of the peptide containing amino acids 45–48, with an iodoacetamide adduct indicating reduced C45 (top) or with a NEM adduct after DTT reduction of a disulfide bond (bottom). Characterization of C106: MS-MS trace of the peptide containing amino acids 97–112 with an iodoacetamide adduct indicating reduced C106 (top); the MS-MS trace from disulfide-HMGB1 was identical and is not depicted. (B) Electrophoretic mobility of recombinant HMGB1 prepared in the presence (+DTT) or absence of DTT (−DTT). Samples were heated in the presence (+) or absence (−) of 350 mM β-mercaptoethanol (β-me), loaded onto a 12% SDS-PA gel, and revealed by Coomassie staining (left) or by Western blotting using a mAb or a pAb against HMGB1 (right). (C) Nuclear translocation of p65-GFP (indicated with white arrows) was visualized by confocal microscopy in living mouse embryonic fibroblasts exposed for 30 min to 80 nM disulfide- or all-thiol HMGB1. Data are representative of three independent experiments. (D) Human macrophages were exposed for 4 h to 0.4 µM of disulfide- or all-thiol-HMGB1. The levels of TNF, IL-6, and IL-8 mRNAs were measured by real-time PCR and expressed as fold increase compared with unstimulated macrophages (*, P < 0.05, Student’s t test). Error bars represent standard deviation. Data are representative of three experiments performed with macrophages from unrelated healthy individuals.
Figure 1. Characterization of HMGB1 redox states. (A) Mass spectrometric characterization of HMGB1 purified with DTT (top) or without DTT (bottom). Characterization of C23: MS-MS trace of the peptide containing amino acids 13–24, with an iodoacetamide adduct indicating reduced C23 (top) or with a NEM adduct after DTT reduction of a disulfide bond (bottom). Characterization of C45: MS-MS trace of the peptide containing amino acids 45–48, with an iodoacetamide adduct indicating reduced C45 (top) or with a NEM adduct after DTT reduction of a disulfide bond (bottom). Characterization of C106: MS-MS trace of the peptide containing amino acids 97–112 with an iodoacetamide adduct indicating reduced C106 (top); the MS-MS trace from disulfide-HMGB1 was identical and is not depicted. (B) Electrophoretic mobility of recombinant HMGB1 prepared in the presence (+DTT) or absence of DTT (−DTT). Samples were heated in the presence (+) or absence (−) of 350 mM β-mercaptoethanol (β-me), loaded onto a 12% SDS-PA gel, and revealed by Coomassie staining (left) or by Western blotting using a mAb or a pAb against HMGB1 (right). (C) Nuclear translocation of p65-GFP (indicated with white arrows) was visualized by confocal microscopy in living mouse embryonic fibroblasts exposed for 30 min to 80 nM disulfide- or all-thiol HMGB1. Data are representative of three independent experiments. (D) Human macrophages were exposed for 4 h to 0.4 µM of disulfide- or all-thiol-HMGB1. The levels of TNF, IL-6, and IL-8 mRNAs were measured by real-time PCR and expressed as fold increase compared with unstimulated macrophages (*, P < 0.05, Student’s t test). Error bars represent standard deviation. Data are representative of three experiments performed with macrophages from unrelated healthy individuals.CC BY-NC-SA · PubMed Central
Figure 2. The cytokine-stimulating and chemoattractant activities of HMGB1 are mutually exclusive. (A) HMGB1/CXCL12 heterocomplex detected by hybrid ELISA. All-thiol- or disulfide-HMGB1 (7.5 ng) were preincubated with the indicated amount CXCL12 at 37°C for 15 min. The formation of the heterocomplex was detected by hybrid ELISA (an anti-CXCL12 capture antibody and an anti-HMGB1 detection antibody). Results are expressed as absorbance at 450 nm (*, P < 0.05, ANOVA). (B) Human monocyte migration in response to increasing concentrations of CXCL12 in the presence or absence of 300 nM all-thiol- or disulfide-HMGB1 (*, P < 0.01 vs. CXCL12 alone; 2-way ANOVA). (C) Migration of mouse 3T3 fibroblasts toward disulfide-HMGB1 or all-thiol-HMGB1, or disufide-HMGB1 exposed to 5 mM of DTT for 30 min (newly all-thiol-HMGB1; *, P < 0.05 vs. disulfide-HMGB1, ANOVA). (D) Migration of 3T3 fibroblasts toward all-thiol-HMGB1 in the presence of increasing concentrations of disulfide-HMGB1, and expression of TNF (as fold increase compared with unstimulated macrophages) in human macrophages stimulated for 4 h with disulfide-HMGB1 in the presence of increasing concentrations of all-thiol-HMGB1. The effects of the competing form of HMGB1 are not statistically significant (ANOVA). (E and F) Migration of mouse 3T3 fibroblasts toward WT all-thiol-HMGB1 previously exposed to increasing concentrations of H2O2 for 1 h (E; *, P < 0.05 vs. all-thiol HMGB1 not treated with H2O2, ANOVA), and toward WT all-thiol-HMGB1 or the E106 mutant purified in the presence of DTT (F; *, P < 0.05 vs. untreated control). (G) Human macrophages were stimulated for 4 h with WT disulfide-HMGB1 or the E106 mutant (0.4 µM) prepared in the absence of DTT. Expression of TNF, MIP-2, and IL-8 was measured by real-time PCR and expressed as fold increase compared with unstimulated macrophages (*, P < 0.05 vs. disulfide-HMGB1; Student’s t test). In all panels, data are representative of at least three independent experiments and bars represent the mean ± SD of triplicate samples (when not visible, they fall within symbols).
Figure 2. The cytokine-stimulating and chemoattractant activities of HMGB1 are mutually exclusive. (A) HMGB1/CXCL12 heterocomplex detected by hybrid ELISA. All-thiol- or disulfide-HMGB1 (7.5 ng) were preincubated with the indicated amount CXCL12 at 37°C for 15 min. The formation of the heterocomplex was detected by hybrid ELISA (an anti-CXCL12 capture antibody and an anti-HMGB1 detection antibody). Results are expressed as absorbance at 450 nm (*, P < 0.05, ANOVA). (B) Human monocyte migration in response to increasing concentrations of CXCL12 in the presence or absence of 300 nM all-thiol- or disulfide-HMGB1 (*, P < 0.01 vs. CXCL12 alone; 2-way ANOVA). (C) Migration of mouse 3T3 fibroblasts toward disulfide-HMGB1 or all-thiol-HMGB1, or disufide-HMGB1 exposed to 5 mM of DTT for 30 min (newly all-thiol-HMGB1; *, P < 0.05 vs. disulfide-HMGB1, ANOVA). (D) Migration of 3T3 fibroblasts toward all-thiol-HMGB1 in the presence of increasing concentrations of disulfide-HMGB1, and expression of TNF (as fold increase compared with unstimulated macrophages) in human macrophages stimulated for 4 h with disulfide-HMGB1 in the presence of increasing concentrations of all-thiol-HMGB1. The effects of the competing form of HMGB1 are not statistically significant (ANOVA). (E and F) Migration of mouse 3T3 fibroblasts toward WT all-thiol-HMGB1 previously exposed to increasing concentrations of H2O2 for 1 h (E; *, P < 0.05 vs. all-thiol HMGB1 not treated with H2O2, ANOVA), and toward WT all-thiol-HMGB1 or the E106 mutant purified in the presence of DTT (F; *, P < 0.05 vs. untreated control). (G) Human macrophages were stimulated for 4 h with WT disulfide-HMGB1 or the E106 mutant (0.4 µM) prepared in the absence of DTT. Expression of TNF, MIP-2, and IL-8 was measured by real-time PCR and expressed as fold increase compared with unstimulated macrophages (*, P < 0.05 vs. disulfide-HMGB1; Student’s t test). In all panels, data are representative of at least three independent experiments and bars represent the mean ± SD of triplicate samples (when not visible, they fall within symbols).CC BY-NC-SA · PubMed Central
Figure 3. BoxA and monoclonal antibody DPH1.1 prevent HMGB1-induced cell migration but not cytokine expression. (A and C) Migration of mouse 3T3 fibroblasts toward WT all-thiol-HMGB1 in the presence or not of BoxA (A) or F(ab’)2 fragments from the DPH1.1 anti-HMGB1 monoclonal antibody (C). (B and D) Human macrophages were stimulated for 4 h with disulfide-HMGB1 (0.4 µM) in the presence of BoxA (B) or DPH1.1 F(ab’)2 fragments (D). Expression of TNF was measured by real-time PCR and expressed as fold increase compared with unstimulated macrophages. In all panels, data are representative of at least three independent experiments and bars represent the mean ± SD of triplicate samples (*, P < 0.05 vs. control; ANOVA).
Figure 3. BoxA and monoclonal antibody DPH1.1 prevent HMGB1-induced cell migration but not cytokine expression. (A and C) Migration of mouse 3T3 fibroblasts toward WT all-thiol-HMGB1 in the presence or not of BoxA (A) or F(ab’)2 fragments from the DPH1.1 anti-HMGB1 monoclonal antibody (C). (B and D) Human macrophages were stimulated for 4 h with disulfide-HMGB1 (0.4 µM) in the presence of BoxA (B) or DPH1.1 F(ab’)2 fragments (D). Expression of TNF was measured by real-time PCR and expressed as fold increase compared with unstimulated macrophages. In all panels, data are representative of at least three independent experiments and bars represent the mean ± SD of triplicate samples (*, P < 0.05 vs. control; ANOVA).CC BY-NC-SA · PubMed Central
Figure 4. HMGB1 cysteines are essential to promote cytokine/chemokine production, but not for chemotaxis. (A) Electrophoretic mobility of recombinant WT HMGB1 and mutants prepared in the absence of DTT, determined as described in the legend of Fig. 1 B. (B) Human macrophages were stimulated for 4 h with mutants or WT HMGB1 prepared in the absence of DTT (0.4 µM). TNF expression was measured by real-time PCR (*, P < 0.05 vs. control, ANOVA). (C and D) 3T3 fibroblast migration toward 1 nM HMGB1 mutants, exposed or not for 1 h to 5 mM DTT (C) or to 100 mM H2O2 (D). Bars represent the mean ± SD of triplicate samples (*, P < 0.05 vs. control, ANOVA).
Figure 4. HMGB1 cysteines are essential to promote cytokine/chemokine production, but not for chemotaxis. (A) Electrophoretic mobility of recombinant WT HMGB1 and mutants prepared in the absence of DTT, determined as described in the legend of Fig. 1 B. (B) Human macrophages were stimulated for 4 h with mutants or WT HMGB1 prepared in the absence of DTT (0.4 µM). TNF expression was measured by real-time PCR (*, P < 0.05 vs. control, ANOVA). (C and D) 3T3 fibroblast migration toward 1 nM HMGB1 mutants, exposed or not for 1 h to 5 mM DTT (C) or to 100 mM H2O2 (D). Bars represent the mean ± SD of triplicate samples (*, P < 0.05 vs. control, ANOVA).CC BY-NC-SA · PubMed Central
Figure 5. Oxidation modulates the activities of HMGB1 in vivo. (A) Electrophoretic mobility of HMGB1 from tibialis anterior muscles harvested at the indicated times after CTX injection. Samples were heated in the presence (+) or absence (–) of 350 mM β-mercaptoethanol (β-me), loaded onto a 12% SDS-PAGE gel and revealed by Western blotting using a polyclonal antibody against HMGB1. Total lysate of tibialis anterior muscle was added as a control (L). (B–D) 3S-HMGB1 induces leukocyte recruitment in vivo. An air pouch was created in mice by the dorsal subcutaneous injection of air. At day 6, the air pouches were injected with 200 µl of PBS containing 10 pmol CXCL12, 300 pmol HMGB1 (WT or 3S), or both. (C) Alternatively, air pouches were injected with 200 µl of PBS containing or not containing 1 or 10 nmol HMGB1 (WT or 3S; C) or with 1 nmol HMGB1 (WT or 3S) in absence or presence of N-acetylcysteine (NAC; 100 nmol/g; D). After 6 h, cells were collected from the air pouches, stained with anti-Ly6C and anti-CD11b antibodies, and analyzed by flow cytometry (WBCs, white blood cells; *, P < 0.05; **, P < 0.01; ***, P < 0.001, ANOVA plus Dunnett’s posttest).
Figure 5. Oxidation modulates the activities of HMGB1 in vivo. (A) Electrophoretic mobility of HMGB1 from tibialis anterior muscles harvested at the indicated times after CTX injection. Samples were heated in the presence (+) or absence (–) of 350 mM β-mercaptoethanol (β-me), loaded onto a 12% SDS-PAGE gel and revealed by Western blotting using a polyclonal antibody against HMGB1. Total lysate of tibialis anterior muscle was added as a control (L). (B–D) 3S-HMGB1 induces leukocyte recruitment in vivo. An air pouch was created in mice by the dorsal subcutaneous injection of air. At day 6, the air pouches were injected with 200 µl of PBS containing 10 pmol CXCL12, 300 pmol HMGB1 (WT or 3S), or both. (C) Alternatively, air pouches were injected with 200 µl of PBS containing or not containing 1 or 10 nmol HMGB1 (WT or 3S; C) or with 1 nmol HMGB1 (WT or 3S) in absence or presence of N-acetylcysteine (NAC; 100 nmol/g; D). After 6 h, cells were collected from the air pouches, stained with anti-Ly6C and anti-CD11b antibodies, and analyzed by flow cytometry (WBCs, white blood cells; *, P < 0.05; **, P < 0.01; ***, P < 0.001, ANOVA plus Dunnett’s posttest).CC BY-NC-SA · PubMed Central

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Authors
Venereau, E., Casalgrandi, M., Schiraldi, M., Antoine, D. J., Cattaneo, A., De Marchis, F., Liu, J., Antonelli, A., Preti, A., Raeli, L., Shams, S. S., Yang, H., Varani, L., Andersson, U., Tracey, K. J., Bachi, A., Uguccioni, M., & Bianchi, M. E.
Affiliations
  • Division of Genetics and Cell Biology, San Raffaele Scientific Institute, 20132 Milan, Italy 1
  • HMGBiotech Srl, 20133 Milan, Italy 2
  • Institute for Research in Biomedicine (CH)
  • University of Liverpool (GB)
  • IRCCS Ospedale San Raffaele (IT)
  • Vita-Salute San Raffaele University (IT)
  • Feinstein Institute for Medical Research (US)
  • Karolinska University Hospital (SE)
Corresponding author
Bianchi, Marco E.
ORCID
Venue
Journal of Experimental Medicine
Publisher
Rockefeller University Press
Published
2012-08-06
Volume
209
Issue
9
Pages
1519-1528
ISSN
  • 0022-1007 (ISSN-L)
  • 1540-9538
DOI
10.1084/jem.20120189
PMC
PMC3428943
OpenAlex
W2141438911
Semantic Scholar
17d0978fd742005f41f9a10b50083eec9ce6ff3d
Cited by
720
References
29
Citations by year
2026: 48 · 2025: 38 · 2024: 54 · 2023: 46 · 2022: 38 · 2021: 44 · 2020: 56 · 2019: 40 · 2018: 60 · 2017: 58 · 2016: 52 · 2015: 64 · 2014: 66 · 2013: 46 · 2012: 10
FWCI
38.20
Type
journal article
Language
en
Open access
Locations
  • The Journal of Experimental Medicine (publishedVersion)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc-sa)
  • PubMed Central (publishedVersion, cc-by-nc-sa)
  • PubMed (publishedVersion)
  • reroDoc Digital Library (submittedVersion)
Funders
  • European Commission (281608, 280873)
  • National Institute of General Medical Sciences (R01GM062508)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Clinical Biochemistry › Advanced Glycation End Products research
  • Life Sciences › Immunology and Microbiology › Immunology › Immune Response and Inflammation
  • Life Sciences › Immunology and Microbiology › Immunology › Neutrophil, Myeloperoxidase and Oxidative Mechanisms
Keywords
HMGB1, high mobility group box 1, redox regulation, cysteine oxidation, disulfide bonds, leukocyte recruitment, sterile inflammation, tissue damage, muscle damage, reactive oxygen species, chemoattractants, leukocyte activation
MeSH terms
Animals, Antibodies, Monoclonal, Cell Movement, Chemotactic Factors, Cysteine, Disulfides, Inflammation, Leukocytes, Male, Mice, Inbred C57BL, Mutation, Recombinant Proteins, Cytokines, Reactive Oxygen Species, Muscle, Skeletal, HMGB1 Protein, Mice, Rats

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