title: Alternatively Activated (M2) Macrophage Phenotype Is Inducible by Endothelin-1 in Cultured Human Macrophages.
abstract: BACKGROUND
  Alternatively activated (M2) macrophages are phenotypically characterized by the expression of specific markers, mainly macrophage scavenger receptors (CD204 and CD163) and mannose receptor-1 (CD206), and participate in the fibrotic process by over-producing pro-fibrotic molecules, such as transforming growth factor-beta1 (TGFbeta1) and metalloproteinase (MMP)-9. Endothelin-1 (ET-1) is implicated in the fibrotic process, exerting its pro-fibrotic effects through the interaction with its receptors (ETA and ETB). The study investigated the possible role of ET-1 in inducing the transition from cultured human macrophages into M2 cells.
  
  
  METHODS
  Cultured human monocytes (THP-1 cell line) were activated into macrophages (M0 macrophages) with phorbol myristate acetate and subsequently maintained in growth medium (M0-controls) or treated with either ET-1 (100nM) or interleukin-4 (IL-4, 10ng/mL, M2 inducer) for 72 hours. Similarly, primary cultures of human peripheral blood monocyte (PBM)-derived macrophages obtained from healthy subjects, were maintained in growth medium (untreated cells) or treated with ET-1 or IL-4 for 6 days. Both M0 and PBM-derived macrophages were pre-treated with ET receptor antagonist (ETA/BRA, bosentan 10-5M) for 1 hour before ET-1 stimulation. Protein and gene expression of CD204, CD206, CD163, TGFbeta1 were analysed by immunocytochemistry, Western blotting and quantitative real time polymerase chain reaction (qRT-PCR). Gene expression of interleukin(IL)-10 and macrophage derived chemokine (CCL-22) was evaluated by qRT-PCR. MMP-9 production was investigated by gel zymography.
  
  
  RESULTS
  ET-1 significantly increased the expression of M2 phenotype markers CD204, CD206, CD163, IL-10 and CCL-22, and the production of MMP-9 in both cultures of M0 and PBM-derived macrophages compared to M0-controls and untreated cells. In cultured PBM-derived macrophages, ET-1 increased TGFbeta1 protein and gene expression compared to untreated cells. The ET-1-mediated effects were contrasted by ETA/BRA treatment in both cultured cell types.
  
  
  CONCLUSIONS
  ET-1 seems to induce the M2 phenotype in cultured human macrophages, a process apparently contrasted by the action of the ETA/BRA, suggesting possible clinical implications in those fibrotic diseases characterized by increased ET-1 concentrations, such as systemic sclerosis but also type 2 diabetes.
authors.affiliation: Research Laboratory and Academic Division of Clinical Rheumatology, Department of Internal Medicine, University of Genova, Genoa, Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: Soldano
authors.affiliation: Research Laboratory and Academic Division of Clinical Rheumatology, Department of Internal Medicine, University of Genova, Genoa, Italy.
authors.forename: Carmen
authors.initials: C
authors.lastname: Pizzorni
authors.affiliation: Research Laboratory and Academic Division of Clinical Rheumatology, Department of Internal Medicine, University of Genova, Genoa, Italy.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Paolino
authors.affiliation: Research Laboratory and Academic Division of Clinical Rheumatology, Department of Internal Medicine, University of Genova, Genoa, Italy.
authors.forename: Amelia Chiara
authors.initials: AC
authors.lastname: Trombetta
authors.affiliation: Research Laboratory and Academic Division of Clinical Rheumatology, Department of Internal Medicine, University of Genova, Genoa, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Montagna
authors.affiliation: Research Laboratory and Academic Division of Clinical Rheumatology, Department of Internal Medicine, University of Genova, Genoa, Italy.
authors.forename: Renata
authors.initials: R
authors.lastname: Brizzolara
authors.affiliation: Research Laboratory and Academic Division of Clinical Rheumatology, Department of Internal Medicine, University of Genova, Genoa, Italy.
authors.forename: Barbara
authors.initials: B
authors.lastname: Ruaro
authors.affiliation: Research Laboratory and Academic Division of Clinical Rheumatology, Department of Internal Medicine, University of Genova, Genoa, Italy.
authors.forename: Alberto
authors.initials: A
authors.lastname: Sulli
authors.affiliation: Research Laboratory and Academic Division of Clinical Rheumatology, Department of Internal Medicine, University of Genova, Genoa, Italy.
authors.forename: Maurizio
authors.initials: M
authors.lastname: Cutolo
mesh_terms: D015703:Antigens, CD / Q000235:genetics / Q000276:immunology; D015214:Antigens, Differentiation, Myelomonocytic / Q000235:genetics / Q000276:immunology; D000077300:Bosentan; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D054422:Chemokine CCL22 / Q000235:genetics / Q000276:immunology; D065128:Endothelin Receptor Antagonists / Q000494:pharmacology; D019332:Endothelin-1 / Q000494:pharmacology*; D005786:Gene Expression Regulation; D006801:Humans; D016753:Interleukin-10 / Q000235:genetics / Q000276:immunology; D015847:Interleukin-4 / Q000494:pharmacology; D037181:Lectins, C-Type / Q000235:genetics / Q000276:immunology; D008262:Macrophage Activation / Q000187:drug effects*; D008264:Macrophages / Q000166:cytology / Q000187:drug effects* / Q000276:immunology; D000090323:Mannose Receptor; D037241:Mannose-Binding Lectins / Q000235:genetics / Q000276:immunology; D020780:Matrix Metalloproteinase 9 / Q000235:genetics / Q000276:immunology; D009000:Monocytes / Q000166:cytology / Q000187:drug effects* / Q000276:immunology; D010641:Phenotype; D061251:Primary Cell Culture; D044022:Receptor, Endothelin A / Q000235:genetics* / Q000276:immunology; D011956:Receptors, Cell Surface / Q000235:genetics / Q000276:immunology; D051117:Scavenger Receptors, Class A / Q000235:genetics / Q000276:immunology; D013449:Sulfonamides / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D053773:Transforming Growth Factor beta1 / Q000235:genetics / Q000276:immunology; D000098902:CD163 Antigen
chemical_list: D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; C515482:CCL22 protein, human; D000098902:CD163 Antigen; D054422:Chemokine CCL22; D065128:Endothelin Receptor Antagonists; D019332:Endothelin-1; C508609:IL10 protein, human; C508598:IL4 protein, human; D037181:Lectins, C-Type; C495139:MSR1 protein, human; D000090323:Mannose Receptor; D037241:Mannose-Binding Lectins; D044022:Receptor, Endothelin A; D011956:Receptors, Cell Surface; D051117:Scavenger Receptors, Class A; D013449:Sulfonamides; C508960:TGFB1 protein, human; D053773:Transforming Growth Factor beta1; D016753:Interleukin-10; D015847:Interleukin-4; C579270:MMP9 protein, human; D020780:Matrix Metalloproteinase 9; D013755:Tetradecanoylphorbol Acetate; D000077300:Bosentan