title: Macrophage migration inhibitory factor (MIF) is rendered enzymatically inactive by myeloperoxidase-derived oxidants but retains its immunomodulatory function.
abstract: Macrophage migration inhibitory factor (MIF) is an important player in the regulation of the inflammatory response. Elevated plasma MIF is found in sepsis, arthritis, cystic fibrosis and atherosclerosis. Immunomodulatory activities of MIF include the ability to promote survival and recruitment of inflammatory cells and to amplify pro-inflammatory cytokine production. MIF has an unusual nucleophilic N-terminal proline with catalytic tautomerase activity. It remains unclear whether tautomerase activity is required for MIF function, but small molecules that inhibit tautomerase activity also inhibit the pro-inflammatory activities of MIF. A prominent feature of the acute inflammatory response is neutrophil activation and production of reactive oxygen species, including myeloperoxidase (MPO)-derived hypochlorous acid and hypothiocyanous acid. We hypothesized that MPO-derived oxidants would oxidize the N-terminal proline of MIF and alter its biological activity. MIF was exposed to hypochlorous acid and hypothiocyanous acid and the oxidative modifications on MIF were examined by LC-MS/MS. Imine formation and carbamylation was observed on the N-terminal proline in response to MPO-dependent generation of hypochlorous and hypothiocyanous acid, respectively. These modifications led to a complete loss of tautomerase activity. However, modified MIF still increased CXCL-8/IL-8 production by peripheral blood mononuclear cells (PBMCs) and blocked neutrophil apoptosis, indicating that tautomerase activity is not essential for these biological functions. Pre-treatment of MIF with hypochlorous acid protected the protein from covalent modification by the MIF inhibitor 4-iodo-6-phenylpyrimidine (4-IPP). Therefore, oxidant generation at inflammatory sites may protect MIF from inactivation by more disruptive electrophiles, including drugs designed to target the tautomerase activity of MIF.
authors.affiliation: Centre for Free Radical Research, Department of Pathology, University of Otago Christchurch, Christchurch, New Zealand. Electronic address: nina.dickerhof@otago.ac.nz.
authors.forename: Nina
authors.initials: N
authors.lastname: Dickerhof
authors.affiliation: Institute of Biochemistry and Molecular Cell Biology, RWTH Aachen University, Aachen, Germany.
authors.forename: Lisa
authors.initials: L
authors.lastname: Schindler
authors.affiliation: Institute of Biochemistry and Molecular Cell Biology, RWTH Aachen University, Aachen, Germany.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Bernhagen
authors.affiliation: Centre for Free Radical Research, Department of Pathology, University of Otago Christchurch, Christchurch, New Zealand.
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Kettle
authors.affiliation: Centre for Free Radical Research, Department of Pathology, University of Otago Christchurch, Christchurch, New Zealand.
authors.forename: Mark B
authors.initials: MB
authors.lastname: Hampton
mesh_terms: D017209:Apoptosis / Q000276:immunology*; D002853:Chromatography, Liquid; D006801:Humans; D007249:Inflammation / Q000378:metabolism*; D016209:Interleukin-8 / Q000096:biosynthesis; D019746:Intramolecular Oxidoreductases / Q000276:immunology / Q000378:metabolism*; D007963:Leukocytes, Mononuclear / Q000276:immunology / Q000378:metabolism; D008263:Macrophage Migration-Inhibitory Factors / Q000276:immunology / Q000378:metabolism*; D009504:Neutrophils / Q000276:immunology / Q000378:metabolism; D016877:Oxidants / Q000378:metabolism; D010084:Oxidation-Reduction; D009195:Peroxidase / Q000378:metabolism*; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: C508606:CXCL8 protein, human; D016209:Interleukin-8; D008263:Macrophage Migration-Inhibitory Factors; D016877:Oxidants; D011994:Recombinant Proteins; D009195:Peroxidase; D019746:Intramolecular Oxidoreductases; C515598:MIF protein, human
keywords: Apoptosis; Carbamylation; Cytokine; D-dopachrome tautomerase (D-DT, MIF-2); Hypochlorous acid; Inflammation; Macrophage migration inhibitory factor (MIF); Myeloperoxidase (MPO); Neutrophil