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Cited by 156Liver physiology and pathologyCytokine Signaling Pathways and InteractionsNF-κB Signaling Pathways

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Hepatocyte Growth Factor Modulates Interleukin-6 Production in Bone Marrow Derived Macrophages: Implications for Inflammatory Mediated Diseases

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Coudriet, Gina M., He, Jing, Trucco, Massimo, Mars, Wendy M., Piganelli, Jon D.·2010·PLoS ONE
DOI 10.1371/journal.pone.0015384PMC PMC2970559
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Abstract

The generation of the pro-inflammatory cytokines IL-6, TNF-α, and IL-1β fuel the acute phase response (APR). To maintain body homeostasis, the increase of inflammatory proteins is resolved by acute phase proteins via presently unknown mechanisms. Hepatocyte growth factor (HGF) is transcribed in response to IL-6. Since IL-6 production promotes the generation of HGF and induces the APR, we posited that accumulating HGF might be a likely candidate for quelling excess inflammation under non-pathological conditions. We sought to assess the role of HGF and how it influences the regulation of inflammation utilizing a well-defined model of inflammatory activation, lipopolysaccharide (LPS)-stimulation of bone marrow derived macrophages (BMM). BMM were isolated from C57BL6 mice and were stimulated with LPS in the presence or absence of HGF. When HGF was present, there was a decrease in production of the pro-inflammatory cytokine IL-6, along with an increase in the anti-inflammatory cytokine IL-10. Altered cytokine production correlated with an increase in phosphorylated GSK3β, increased retention of the phosphorylated NFκB p65 subunit in the cytoplasm, and an enhanced interaction between CBP and phospho-CREB. These changes were a direct result of signaling through the HGF receptor, MET, as effects were reversed in the presence of a selective inhibitor of MET (SU11274) or when using BMM from macrophage-specific conditional MET knockout mice. Combined, these data provide compelling evidence that under normal circumstances, HGF acts to suppress the inflammatory response.

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Figure 1 HGF modulates IL-6 production in LPS stimulated macrophages. BMM derived from C57BL6 mice were pretreated with or without 10 pg and 10 ng HGF for 24 hours and stimulated with 1 µg/ml LPS. Cell culture media was collected (24 h) and IL-6 levels were measured by ELISA. Results are representative of the mean (± SEM) of three independent experiment done in triplicate, * indicates <0.001.
Figure 1 HGF modulates IL-6 production in LPS stimulated macrophages. BMM derived from C57BL6 mice were pretreated with or without 10 pg and 10 ng HGF for 24 hours and stimulated with 1 µg/ml LPS. Cell culture media was collected (24 h) and IL-6 levels were measured by ELISA. Results are representative of the mean (± SEM) of three independent experiment done in triplicate, * indicates <0.001.CC BY · PubMed Central
Figure 2 A MET kinase inhibitor abrogates HGF suppression of IL-6. Using an optimal dose of the MET inhibitor SU11274 (1 µg), BMM were pretreated for 2 hours before an overnight incubation with 10 and 100 pg HGF and 24 hour stimulation with LPS. Results are representative of the mean (± SEM) of three independent experiments done in triplicate. *, p = 0.02 vs. the respective control group.
Figure 2 A MET kinase inhibitor abrogates HGF suppression of IL-6. Using an optimal dose of the MET inhibitor SU11274 (1 µg), BMM were pretreated for 2 hours before an overnight incubation with 10 and 100 pg HGF and 24 hour stimulation with LPS. Results are representative of the mean (± SEM) of three independent experiments done in triplicate. *, p = 0.02 vs. the respective control group.CC BY · PubMed Central
Figure 3 Deletion of the HGF receptor MET demonstrates a reversal in the effects of HGF on LPS stimulated BMM. BMM derived from either macrophage specific MET conditional knockout mice (METfl/fl:crelysZ+/−) or their wild type littermate controls (METfl/fl:crelysZ−/−) were pretreated with or without 1, 10 and 100 pg HGF for 24 hours and stimulated with 1 µg/ml LPS. Cell culture media was collected (24 h) and IL-6 levels were measured by ELISA. Results are representative of two independent experiments done in triplicate.
Figure 3 Deletion of the HGF receptor MET demonstrates a reversal in the effects of HGF on LPS stimulated BMM. BMM derived from either macrophage specific MET conditional knockout mice (METfl/fl:crelysZ+/−) or their wild type littermate controls (METfl/fl:crelysZ−/−) were pretreated with or without 1, 10 and 100 pg HGF for 24 hours and stimulated with 1 µg/ml LPS. Cell culture media was collected (24 h) and IL-6 levels were measured by ELISA. Results are representative of two independent experiments done in triplicate.CC BY · PubMed Central
Figure 4 Treatment with HGF leads to increased GSK3β phosphorylation. (A) Cytoplasmic lysates prepared from BMM were incubated with 10 ng HGF for 24 hours prior to stimulation with LPS (1 µg/ml) for 15 minutes. The lysates were separated by SDS-PAGE and probed with a phospho-specific GSK3β antibody before re-probing for β-actin. (B) Densitometric analysis for phospho-GSK3β fold induction normalized to β-actin is shown for 3 separate experiments. Note that all experiments show induction greater than 1 when HGF is present.
Figure 4 Treatment with HGF leads to increased GSK3β phosphorylation. (A) Cytoplasmic lysates prepared from BMM were incubated with 10 ng HGF for 24 hours prior to stimulation with LPS (1 µg/ml) for 15 minutes. The lysates were separated by SDS-PAGE and probed with a phospho-specific GSK3β antibody before re-probing for β-actin. (B) Densitometric analysis for phospho-GSK3β fold induction normalized to β-actin is shown for 3 separate experiments. Note that all experiments show induction greater than 1 when HGF is present.CC BY · PubMed Central
Figure 5 HGF promotes the interaction of phosphorylated CREB with CBP along with an increased production of IL-10. (A) Whole cell lysates prepared from BMM were incubated overnight with HGF (10 ng) prior to stimulation LPS (1 µg/ml) for 15 minutes, then subjected to immunoprecipitation with a CBP antibody. The lysates were separated by SDS-PAGE and probed with a phospho-specific CREB antibody before re-probing for β-actin. (B) Densitometric analysis for phospho-CREB fold induction normalized to β-actin. Is shown for 3 separate experiments. Note that all experiments show induction greater than 1 when HGF is present. (C) BMM were pretreated with or without 1 µM of the MET kinase inhibitor (SU11274) for 2 h prior to incubation with HGF (1, 10 and 100 pg) for 24 hours followed by stimulation with 1 µg/ml LPS. Cell culture media was collected (24 h) and IL-10 levels were measured by ELISA. Results are representative of two independent experiments done in triplicate.
Figure 5 HGF promotes the interaction of phosphorylated CREB with CBP along with an increased production of IL-10. (A) Whole cell lysates prepared from BMM were incubated overnight with HGF (10 ng) prior to stimulation LPS (1 µg/ml) for 15 minutes, then subjected to immunoprecipitation with a CBP antibody. The lysates were separated by SDS-PAGE and probed with a phospho-specific CREB antibody before re-probing for β-actin. (B) Densitometric analysis for phospho-CREB fold induction normalized to β-actin. Is shown for 3 separate experiments. Note that all experiments show induction greater than 1 when HGF is present. (C) BMM were pretreated with or without 1 µM of the MET kinase inhibitor (SU11274) for 2 h prior to incubation with HGF (1, 10 and 100 pg) for 24 hours followed by stimulation with 1 µg/ml LPS. Cell culture media was collected (24 h) and IL-10 levels were measured by ELISA. Results are representative of two independent experiments done in triplicate.CC BY · PubMed Central
Figure 6 HGF prevents the nuclear translocation of phosphorylated p65. BMM were (A) untreated (B) stimulated with LPS (1 µg/ml) (C) treated with 10 pg HGF and stimulated with LPS (1 µg/ml) or (D) treated with the MET kinase inhibitor, SU11274, 10 pg HGF and stimulated with LPS (1 µg/ml). Cytospin preparations were then stained by immunoflourescence for phosphorylated p65 (Ser 276) and for nuclei with DAPI. The blue staining indicates nuclei, red staining indicates phosphorylated p65, and purple staining indicates colocalization of phospho-p65 within the nucleus.
Figure 6 HGF prevents the nuclear translocation of phosphorylated p65. BMM were (A) untreated (B) stimulated with LPS (1 µg/ml) (C) treated with 10 pg HGF and stimulated with LPS (1 µg/ml) or (D) treated with the MET kinase inhibitor, SU11274, 10 pg HGF and stimulated with LPS (1 µg/ml). Cytospin preparations were then stained by immunoflourescence for phosphorylated p65 (Ser 276) and for nuclei with DAPI. The blue staining indicates nuclei, red staining indicates phosphorylated p65, and purple staining indicates colocalization of phospho-p65 within the nucleus.CC BY · PubMed Central
Figure 7 Proposed mechanism of HGF-mediated suppression. The canonical signaling pathway of LPS-TLR engagement leads to NFκB dependent pro-inflammatory cytokine production through the interaction of CBP with NFκB. However, TLR signaling has also been shown to weakly activate alternative signaling through PI3K, resulting in phosphorylation and inactivation of GSK3β, subsequent sequestration of CBP from NFκB to phospho-CREB, and resultant anti-inflammatory (IL-10) production[7]. Our results show that the presence of HGF enhances the IL-10 pathway. We postulate that in order to resolve inflammation, the generation of IL-6 by LPS-TLR signaling leads to the production of HGF, ultimately leading to the inhibition of inflammation. Hence, we propose HGF acts as an internal rheostat for resolving acute phase inflammatory responses.
Figure 7 Proposed mechanism of HGF-mediated suppression. The canonical signaling pathway of LPS-TLR engagement leads to NFκB dependent pro-inflammatory cytokine production through the interaction of CBP with NFκB. However, TLR signaling has also been shown to weakly activate alternative signaling through PI3K, resulting in phosphorylation and inactivation of GSK3β, subsequent sequestration of CBP from NFκB to phospho-CREB, and resultant anti-inflammatory (IL-10) production[7]. Our results show that the presence of HGF enhances the IL-10 pathway. We postulate that in order to resolve inflammation, the generation of IL-6 by LPS-TLR signaling leads to the production of HGF, ultimately leading to the inhibition of inflammation. Hence, we propose HGF acts as an internal rheostat for resolving acute phase inflammatory responses.CC BY · PubMed Central

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Metadata

Authors
Coudriet, G. M., He, J., Trucco, M., Mars, W. M., & Piganelli, J. D.
Affiliations
University of Pittsburgh (US)
Corresponding author
Piganelli, Jon D.
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2010-11-02
Volume
5
Issue
11
Pages
e15384
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0015384
PMC
PMC2970559
OpenAlex
W2164624420
Cited by
155
References
39
Citations by year
2026: 2 · 2025: 6 · 2024: 17 · 2023: 6 · 2022: 13 · 2021: 14 · 2020: 12 · 2019: 9 · 2018: 10 · 2017: 8 · 2016: 17 · 2015: 6 · 2014: 11 · 2013: 9 · 2012: 10
FWCI
5.04
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • D-Scholarship@Pitt (University of Pittsburgh) (acceptedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
  • American Diabetes Association (CDA 7.07)
  • American Heart Association
  • University of Pittsburgh
  • Medical Center, University of Pittsburgh
  • Children's Hospital of Pittsburgh
Topics
  • Health Sciences › Medicine › Hepatology › Liver physiology and pathology
  • Life Sciences › Immunology and Microbiology › Immunology › Cytokine Signaling Pathways and Interactions
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › NF-κB Signaling Pathways
Keywords
hepatocyte growth factor, interleukin-6, bone marrow-derived macrophages, lipopolysaccharide stimulation, acute phase response, inflammation resolution, GSK3β, NF-κB p65, CREB, CBP, IL-10, anti-inflammatory cytokines
MeSH terms
Glycogen Synthase Kinase 3 beta, Animals, Bone Marrow Cells, Cells, Cultured, Dose-Response Relationship, Drug, Female, Indoles, Inflammation, Lipopolysaccharides, Macrophages, Male, Membrane Proteins, Mice, Inbred C57BL, Phosphoproteins, Phosphorylation, Piperazines, Protein Binding, Sulfonamides, Blotting, Western, Interleukin-6, Hepatocyte Growth Factor, Cyclic AMP Response Element-Binding Protein, Mice, Knockout, Proto-Oncogene Proteins c-met, Glycogen Synthase Kinase 3, Immunoprecipitation, Mice, Transcription Factor RelA, Mice, 129 Strain

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