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Myeloperoxidase Functions as a Major Enzymatic Catalyst for Initiation of Lipid Peroxidation at Sites of Inflammation

2002; Elsevier BV; Volume: 277; Issue: 48 Linguagem: Inglês

10.1074/jbc.m209124200

ISSN

1083-351X

Autores

Renliang Zhang, Marie-Luise Brennan, Zhongzhou Shen, Jennifer C. MacPherson, Dave Schmitt, Cheryl E. Molenda, Stanley L. Hazen,

Tópico(s)

Vanadium and Halogenation Chemistry

Resumo

Initiation of lipid peroxidation and the formation of bioactive eicosanoids are pivotal processes in inflammation and atherosclerosis. Currently, lipoxygenases, cyclooxygenases, and cytochrome P450 monooxygenases are considered the primary enzymatic participants in these events. Myeloperoxidase (MPO), a heme protein secreted by activated leukocytes, generates reactive intermediates that promote lipid peroxidation in vitro. For example, MPO catalyzes oxidation of tyrosine and nitrite to form tyrosyl radical and nitrogen dioxide (⋅NO2), respectively, reactive intermediates capable of initiating oxidation of lipids in plasma. Neither the ability of MPO to initiate lipid peroxidation in vivo nor its role in generating bioactive eicosanoids during inflammation has been reported. Using a model of inflammation (peritonitis) with MPO knockout mice (MPO−/−), we examined the role for MPO in the formation of bioactive lipid oxidation products and promoting oxidant stressin vivo. Electrospray ionization tandem mass spectrometry was used to simultaneously quantify individual molecular species of hydroxy- and hydroperoxy-eicosatetraenoic acids (H(P)ETEs), F2-isoprostanes, hydroxy- and hydroperoxy-octadecadienoic acids (H(P)ODEs), and their precursors, arachidonic acid and linoleic acid. Peritonitis-triggered formation of F2-isoprostanes, a marker of oxidant stress in vivo, was reduced by 85% in the MPO−/− mice. Similarly, formation of all molecular species of H(P)ETEs and H(P)ODEs monitored were significantly reduced (by at least 50%) in the MPO−/− group during inflammation. Parallel analyses of peritoneal lavage proteins for protein dityrosine and nitrotyrosine, molecular markers for oxidative modification by tyrosyl radical and ⋅NO2, respectively, revealed marked reductions in the content of nitrotyrosine, but not dityrosine, in MPO−/−samples. Thus, MPO serves as a major enzymatic catalyst of lipid peroxidation at sites of inflammation. Moreover, MPO-dependent formation of ⋅NO-derived oxidants, and not tyrosyl radical, appears to serve as a preferred pathway for initiating lipid peroxidation and promoting oxidant stress in vivo. Initiation of lipid peroxidation and the formation of bioactive eicosanoids are pivotal processes in inflammation and atherosclerosis. Currently, lipoxygenases, cyclooxygenases, and cytochrome P450 monooxygenases are considered the primary enzymatic participants in these events. Myeloperoxidase (MPO), a heme protein secreted by activated leukocytes, generates reactive intermediates that promote lipid peroxidation in vitro. For example, MPO catalyzes oxidation of tyrosine and nitrite to form tyrosyl radical and nitrogen dioxide (⋅NO2), respectively, reactive intermediates capable of initiating oxidation of lipids in plasma. Neither the ability of MPO to initiate lipid peroxidation in vivo nor its role in generating bioactive eicosanoids during inflammation has been reported. Using a model of inflammation (peritonitis) with MPO knockout mice (MPO−/−), we examined the role for MPO in the formation of bioactive lipid oxidation products and promoting oxidant stressin vivo. Electrospray ionization tandem mass spectrometry was used to simultaneously quantify individual molecular species of hydroxy- and hydroperoxy-eicosatetraenoic acids (H(P)ETEs), F2-isoprostanes, hydroxy- and hydroperoxy-octadecadienoic acids (H(P)ODEs), and their precursors, arachidonic acid and linoleic acid. Peritonitis-triggered formation of F2-isoprostanes, a marker of oxidant stress in vivo, was reduced by 85% in the MPO−/− mice. Similarly, formation of all molecular species of H(P)ETEs and H(P)ODEs monitored were significantly reduced (by at least 50%) in the MPO−/− group during inflammation. Parallel analyses of peritoneal lavage proteins for protein dityrosine and nitrotyrosine, molecular markers for oxidative modification by tyrosyl radical and ⋅NO2, respectively, revealed marked reductions in the content of nitrotyrosine, but not dityrosine, in MPO−/−samples. Thus, MPO serves as a major enzymatic catalyst of lipid peroxidation at sites of inflammation. Moreover, MPO-dependent formation of ⋅NO-derived oxidants, and not tyrosyl radical, appears to serve as a preferred pathway for initiating lipid peroxidation and promoting oxidant stress in vivo. A characteristic feature of inflammation is the concomitant peroxidation of lipids and formation of bioactive lipid peroxidation products (1Yla-Herttuala S. Ann. N. Y. Acad. Sci. 1999; 874: 134-137Crossref PubMed Scopus (115) Google Scholar, 2Chisolm III, G.M. Hazen S.L. Fox P.L. Cathcart M.K. J. Biol. Chem. 1999; 274: 25959-25962Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 3Podrez E.A. Abu-Soud H.M. 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Lipoxygenases, cyclooxygenases, and cytochrome P450 mono-oxygenases are widely thought to serve as the major enzymatic pathways involved (2Chisolm III, G.M. Hazen S.L. Fox P.L. Cathcart M.K. J. Biol. Chem. 1999; 274: 25959-25962Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 6Funk C.D. Science. 2001; 294: 1871-1875Crossref PubMed Scopus (3030) Google Scholar, 14Kuehl Jr., F.A. Egan R.W. Science. 1980; 210: 978-984Crossref PubMed Scopus (547) Google Scholar, 15Needleman P. Turk J. Jakschik B.A. Morrison A.R. Lefkowith J.B. Annu. Rev. Biochem. 1986; 55: 69-102Crossref PubMed Google Scholar, 16Lewis R.A. Austen K.F. Soberman R.J. N. Engl. J. Med. 1990; 323: 645-655Crossref PubMed Scopus (1179) Google Scholar, 17Cathcart M.K. Folcik V.A. Free Radic. Biol. Med. 2000; 28: 1726-1734Crossref PubMed Scopus (98) Google Scholar). These enzymes are expressed in leukocytes and catalyze the direct insertion of molecular oxygen into polyenoic fatty acids, forming hydroperoxides, which are both rapidly reduced to their corresponding alcohols and converted into more advanced oxidation products such as prostaglandins and leukotrienes. For example, hydroperoxy-eicosatetraenoic acids (HPETEs) 1The abbreviations used for: H(P)ETEs, hydroxy-eicosatetraenoic acid and hydroperoxy-eicosatetraenoic acids; H(P)ODEs, hydroxy-octadecadienoic acid and hydroperoxy-octadecadienoic acids; HETEs, hydroxy-eicosatetraenoic acids; HODEs, hydroxy-octadecadienoic acids; COX, cyclooxygenase; DTPA, diethylenetriamine pentaacetic acid; HPLC, high performance liquid chromatography; LC/ESI/MS/MS, reverse phase HPLC with on-line electrospray ionization tandem mass spectrometry; LO, lipoxygenase; MPO, myeloperoxidase; ⋅NO, nitric oxide (nitrogen monoxide); O 2⨪, superoxide; Tg, thioglycollate; Z, zymosan; 12-HETE-d812, (S)-hydroxy-5,8,10,14-eicosatetraenoic-5,6,8,9,11, 12,14,15-d8 acid; PGF2α-d4, prostaglandin F2α; WT, wild type. 1The abbreviations used for: H(P)ETEs, hydroxy-eicosatetraenoic acid and hydroperoxy-eicosatetraenoic acids; H(P)ODEs, hydroxy-octadecadienoic acid and hydroperoxy-octadecadienoic acids; HETEs, hydroxy-eicosatetraenoic acids; HODEs, hydroxy-octadecadienoic acids; COX, cyclooxygenase; DTPA, diethylenetriamine pentaacetic acid; HPLC, high performance liquid chromatography; LC/ESI/MS/MS, reverse phase HPLC with on-line electrospray ionization tandem mass spectrometry; LO, lipoxygenase; MPO, myeloperoxidase; ⋅NO, nitric oxide (nitrogen monoxide); O 2⨪, superoxide; Tg, thioglycollate; Z, zymosan; 12-HETE-d812, (S)-hydroxy-5,8,10,14-eicosatetraenoic-5,6,8,9,11, 12,14,15-d8 acid; PGF2α-d4, prostaglandin F2α; WT, wild type. and their corresponding reduced forms, hydroxyeicosatetraenoic acids (HETEs), are initial products of arachidonic acid metabolism (extensively reviewed in Ref. 18Spector A.A. Gordon J.A. Moore S.A. Prog. Lipid Res. 1988; 27: 271-323Crossref PubMed Scopus (261) Google Scholar). These species have potent pro-inflammatory actions and serve as precursors for numerous eicosanoids (18Spector A.A. Gordon J.A. Moore S.A. Prog. Lipid Res. 1988; 27: 271-323Crossref PubMed Scopus (261) Google Scholar). Their formation has predominantly been attributed to lipoxygenase pathways, and to a lesser extent, cyclooxygenase and cytochrome P450 monooxygenase pathways (2Chisolm III, G.M. Hazen S.L. Fox P.L. Cathcart M.K. J. Biol. Chem. 1999; 274: 25959-25962Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar,14Kuehl Jr., F.A. Egan R.W. Science. 1980; 210: 978-984Crossref PubMed Scopus (547) Google Scholar, 15Needleman P. Turk J. Jakschik B.A. Morrison A.R. Lefkowith J.B. Annu. Rev. Biochem. 1986; 55: 69-102Crossref PubMed Google Scholar, 16Lewis R.A. Austen K.F. Soberman R.J. N. Engl. J. Med. 1990; 323: 645-655Crossref PubMed Scopus (1179) Google Scholar). 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It has also been proposed that MPO functions not to generate biologically active lipids but rather to destroy them through oxidative mechanisms, thereby altering inflammatory responses (49Henderson W.R. Klebanoff S.J. J. Biol. Chem. 1983; 258: 13522-13527Abstract Full Text PDF PubMed Google Scholar). Thus, the role of MPO in initiation of lipid peroxidation in vivo is uncertain, and only through direct experimental investigation will the contribution of MPO to this process be enumerated. Here we employ a combination of murine models of inflammation and quantification of specific lipid oxidation products to define the role of MPO in the initiation of lipid peroxidation in vivo. Further, through parallel analyses of protein molecular markers of distinct oxidation pathways, the relative contributions of MPO-generated tyrosyl radical and reactive nitrogen species pathways to lipid peroxidation are assessed. Free fatty acids were purchased from Cayman Chemical Company (Ann Arbor, MI). Organic solvents were obtained from Fisher Scientific Co. (Pittsburgh, PA). All other reagents were purchased from Sigma unless otherwise indicated. All buffers were passed over a Chelex-100 resin column (Bio-Rad) and supplemented with 0.1 mm diethylenetriamine pentaacetic acid (DTPA) to remove potential contaminant transition metal ions that might catalyze lipid oxidation. Protein content was determined using Bradford-based Bio-Rad protein assay using IgG as protein standard. Wild type (MPO+/+) and MPO−/− mice were on a C57BL/6J background (>98% genetic homogeneity). Age- and sex-matched MPO+/+ and MPO−/− mice were used for all studies. Superoxide and HOCl generation in recovered peritoneal lavage cells from MPO+/+ and MPO−/− mice were performed as recently described (50Brennan M.L. Anderson M.M. Shih D.M. Qu X.D. Wang X. Mehta A.C. Lim L.L. Shi W. Hazen S.L. Jacob J.S. Crowley J.R. Heinecke J.W. Lusis A.J. J. Clin. Invest. 2001; 107: 419-430Crossref PubMed Scopus (282) Google Scholar). Nitrotyrosine, chlorotyrosine, and dityrosine were quantified by stable isotope dilution mass spectrometry-based methods as previously described (32Brennan M.L. Wu W. Fu X. Shen Z. Song W. Frost H. Vadseth C. Narine L. Lenkiewicz E. Borchers M.T. Lusis A.J. Lee J.J. Lee N.A. Abu-Soud H.M. Ischiropoulos H. Hazen S.L. J. Biol. Chem. 2002; 277: 17415-17427Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar). All animal studies were performed using approved protocols from the Animal Research Committee of the Cleveland Clinic Foundation. MPO+/+ and MPO−/− mice were injected intraperitoneally with 1 ml of 4% thioglycollate broth. Twenty hours after recruitment, mice were injected with zymosan (250 mg/kg). Peritoneal lavage was performed in control animals (20 h post intraperitoneal injection of normal saline), 20 h following thioglycollate injection, or 24 h following the combination of both thioglycollate injection (20 h) followed by zymosan injection (4 h), as indicated (Tg/Z). Peritoneal lavages were performed with phosphate-buffered saline containing antioxidant (0.1 mmbutylated hydroxytoluene (BHT)) and metal chelator (2 mmDTPA), transferred to screw-capped tubes covered with argon atmosphere, and then immediately centrifuged at 1000 rpm for 10 min at 4 °C. All analyses of protein and lipid peroxidation products were performed on cell-free lavage supernatants. Cell pellets were resuspended in phosphate-buffered saline containing 0.1 mm BHT and 0.1 mm DTPA, and differentials were performed on cytospin preparations of isolated cells. Cell pellets were also used for Western analyses (see below). Both cells and cell-free lavage fluid not immediately analyzed were overlaid with argon, snap frozen in liquid nitrogen, and stored at −80 °C until analysis. Cell pellets recovered from peritoneal lavages were lysed, concentration measured, and 25 μg total protein loaded per lane in 10% Tris-HCl gel. Following SDS-PAGE analysis, proteins were transferred to polyvinylidene difluoride membrane and probed with rabbit antibodies against mouse COX-1, COX-2, and 12-LO (all from Cayman Chemical) and affinity purified rabbit antibody generated against a peptide fragment of actin (Sigma). Detection was performed using secondary anti-rabbit antibodies conjugated with horseradish peroxidase for chemiluminescent detection. Quantification of bands was performed by densitometric analysis. Band intensities were normalized to actin to account for loading variations between lanes. The mean relative band intensity (normalized to actin) from MPO+/+mice were assigned a relative value of 1.0 for comparisons between MPO+/+ and MPO−/− mice. All steps were performed under either argon or nitrogen atmosphere. Peritoneal lavage volume was reduced under vacuum centrifugation prior to extraction and preparation for mass spectrometry analysis. Immediately prior to extraction, 10 ng each of two deuterated internal standards, 12(S)-hydroxy-5,8,10,14-eicosatetraenoic-5,6,8,9,11,12,14,15-d8 acid (12-HETE-d8) and prostaglandin F2α(PGF2α-d4) (Cayman Chemical Company) were added to each sample. Hydroperoxides in samples were then reduced to their corresponding stable alcohols by adding 1 mmSnCl2 (23Zhang R. Shen Z. Nauseef W.M. Hazen S.L. Blood. 2002; 99: 1802-1810Crossref PubMed Google Scholar). Lipids were then extracted by adding a solvent mixture (1 m acetic acid/2-isopropanol/hexane (2:20:30, v/v/v) to the sample at a ratio of 2.5 ml of solvent mixture/1 ml of sample, vortexing, and then adding 2.5 ml hexane. Following vortex and centrifugation, lipids were recovered in the hexane layer. Peritoneal lavages were re-extracted by addition of an equal volume of hexane, followed by vortex and centrifugation. The combined hexane layers were dried under N2 flow and then analyzed following saponification to release all esterified fatty acid oxidation products. For saponification, N2-dried lipids were resuspended in 1.5 ml of 2-isopropanol, and then fatty acids were released by base hydrolysis with 1.5 ml of 0.2 m NaOH at 60 °C for 30 min under argon atmosphere. Hydrolyzed samples were first acidified to pH 3.0 with 0.5 m HCl, and then fatty acids were extracted twice with 4 ml of hexane. The combined hexane layers were dried under N2 flow, resuspended in 100 μl of methanol, and stored under argon at −80 °C until analysis by reverse phase high performance liquid chromatography (HPLC) with on-line electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS), as described below. LC/ESI/MS/MS was employed to quantify the multiple distinct oxidation products of arachidonic acid and linoleic acid, including individual HETEs, F2-isoprostanes, and hydroxy-octadecadienoic acids (HODEs). Immediately prior to analysis, one volume of H2O was added to 20 volumes methanol-suspended sample, which was then passed through a 0.22-μm filter (Millipore Corporation, Bedford, MA). Sample (50 μl) was injected onto a C-18 column (2 × 250 mm, 5 μm ODS) (Phenomenex, Rancho Palos Verdes, CA) at a flow rate of 0.2 ml/min. The separation was performed using a gradient starting from 85% methanol over 15 min, then to 100% methanol over 1 min, followed by 100% methanol for 15 min. HPLC column effluent was split so that only 50% was introduced into a

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