Joseph P. Gaut

Neutrophils employ the myeloperoxidase system to generate antimicrobial brominating and chlorinating oxidants during sepsis

The myeloperoxidase system of neutrophils uses hydrogen peroxide and chloride to generate hypochlorous acid, a potent bactericidal oxidant in vitro. In a mouse model of polymicrobial sepsis, we observed that mice deficient in myeloperoxidase were more likely than wild-type mice to die from infection. Mass spectrometric analysis of peritoneal inflammatory fluid from septic wild-type mice detected elevated concentrations of 3-chlorotyrosine, a characteristic end product of the myeloperoxidase system. Levels of 3-chlorotyrosine did not rise in the septic myeloperoxidase-deficient mice. Thus, myeloperoxidase seems to protect against sepsis in vivo by producing halogenating species. Surprisingly, levels of 3-bromotyrosine also were elevated in peritoneal fluid from septic wild-type mice and were markedly reduced in peritoneal fluid from septic myeloperoxidase-deficient mice. Furthermore, physiologic concentrations of bromide modulated the bactericidal effects of myeloperoxidase in vitro. It seems, therefore, that myeloperoxidase can use bromide as well as chloride to produce oxidants in vivo, even though the extracellular concentration of bromide is at least 1,000-fold lower than that of chloride. Thus, myeloperoxidase plays an important role in host defense against bacterial pathogens, and bromide might be a previously unsuspected component of this system.

Mechanisms of Cardiac and Renal Dysfunction in Patients Dying of Sepsis

RATIONALE The mechanistic basis for cardiac and renal dysfunction in sepsis is unknown. In particular, the degree and type of cell death is undefined. OBJECTIVES To evaluate the degree of sepsis-induced cardiomyocyte and renal tubular cell injury and death. METHODS Light and electron microscopy and immunohistochemical staining for markers of cellular injury and stress, including connexin-43 and kidney-injury-molecule-1 (Kim-1), were used in this study. MEASUREMENTS AND MAIN RESULTS Rapid postmortem cardiac and renal harvest was performed in 44 septic patients. Control hearts were obtained from 12 transplant and 13 brain-dead patients. Control kidneys were obtained from 20 trauma patients and eight patients with cancer. Immunohistochemistry demonstrated low levels of apoptotic cardiomyocytes (<1-2 cells per thousand) in septic and control subjects and revealed redistribution of connexin-43 to lateral membranes in sepsis (P < 0.020). Electron microscopy showed hydropic mitochondria only in septic specimens, whereas mitochondrial membrane injury and autophagolysosomes were present equally in control and septic specimens. Control kidneys appeared relatively normal by light microscopy; 3 of 20 specimens showed focal injury in approximately 1% of renal cortical tubules. Conversely, focal acute tubular injury was present in 78% of septic kidneys, occurring in 10.3 ± 9.5% and 32.3 ± 17.8% of corticomedullary-junction tubules by conventional light microscopy and Kim-1 immunostains, respectively (P < 0.01). Electron microscopy revealed increased tubular injury in sepsis, including hydropic mitochondria and increased autophagosomes. CONCLUSIONS Cell death is rare in sepsis-induced cardiac dysfunction, but cardiomyocyte injury occurs. Renal tubular injury is common in sepsis but presents focally; most renal tubular cells appear normal. The degree of cell injury and death does not account for severity of sepsis-induced organ dysfunction.

Myeloperoxidase produces nitrating oxidants in vivo

Despite intense interest in pathways that generate reactive nitrogen species, the physiologically relevant mechanisms for inflammatory tissue injury remain poorly understood. One possible mediator is myeloperoxidase, a major constituent of neutrophils, monocytes, and some populations of macrophages. The enzyme uses hydrogen peroxide and nitrite to generate 3-nitrotyrosine in vitro. To determine whether myeloperoxidase produces nitrating intermediates in vivo, we used isotope dilution gas chromatography/mass spectrometry to quantify 3-nitrotyrosine in two models of peritoneal inflammation: mice infected with Klebsiella pneumoniae and mice subjected to cecal ligation and puncture. Both models developed an intense neutrophil inflammatory response, and the inflammatory fluid contained markedly elevated levels of 3-chlorotyrosine, a marker of myeloperoxidase action. In striking contrast, 3-nitrotyrosine levels rose only in the mice infected with K. pneumoniae. Levels of total nitrite and nitrate were 20-fold higher in mice injected with K. pneumoniae than in mice subjected to cecal ligation and puncture. Levels of 3-nitrotyrosine failed to increase in mice infected with K. pneumoniae that lacked functional myeloperoxidase. Our observations provide strong evidence that myeloperoxidase generates reactive nitrogen species in vivo and that it operates in this fashion only when nitrite and nitrate become available.

Acute Antibody-mediated Rejection After Lung Transplantation

BACKGROUND Antibody-mediated rejection (AMR) after lung transplantation remains enigmatic, and there is no consensus on the characteristic clinical, immunologic and histologic features. METHODS We performed a retrospective, single-center cohort study and identified cases of acute AMR based on the presence of circulating donor-specific human leukocyte antigen (HLA) antibodies (DSA), histologic evidence of acute lung injury, C4d deposition and clinical allograft dysfunction. RESULTS We identified 21 recipients with acute AMR based on the aforementioned criteria. AMR occurred a median 258 days after transplantation; 7 recipients developed AMR within 45 days of transplantation. All patients had clinical allograft dysfunction, DSA, histology of acute lung injury and capillary endothelial C4d deposition. Fifteen recipients improved clinically and survived to hospital discharge, but 6 died of refractory AMR. One survivor had bronchiolitis obliterans syndrome at the time of AMR diagnosis; 13 of the 14 remaining survivors developed chronic lung allograft dysfunction (CLAD) during follow-up. Overall, 15 recipients died during the study period, and the median survival after the diagnosis of AMR was 593 days. CONCLUSIONS Acute AMR can be a fulminant form of lung rejection, and survivors are at increased risk of developing CLAD. The constellation of acute lung injury, DSA and capillary endothelial C4d deposition is compelling for acute AMR in recipients with allograft dysfunction. This clinicopathologic definition requires validation in a multicenter cohort, but may serve as a foundation for future studies to further characterize AMR.

Mechanisms for oxidizing low-density lipoprotein. Insights from patterns of oxidation products in the artery wall and from mouse models of atherosclerosis.

The oxidation hypothesis proposes that oxidative modification of low density lipoprotein (LDL) plays a critical role in atherogenesis. This review critically evaluates the various mechanisms proposed for LDL oxidation, focusing on insights derived from chemical analysis of human artery wall and studies of genetically engineered mice. The implications of recent clinical trials of vitamin E for the oxidation hypothesis are also briefly discussed.

Myeloperoxidase Plays Critical Roles in Killing Klebsiella pneumoniae and Inactivating Neutrophil Elastase: Effects on Host Defense

Activated neutrophils use myeloperoxidase (MPO) to generate an array of potent toxic oxidants. In the current studies we used genetically altered mice deficient in MPO to investigate the role of the enzyme in host defense against the Gram-negative bacterium Klebsiella pneumoniae, an important human pathogen. For comparison, we used mice deficient in the antimicrobial molecule, neutrophil elastase (NE). When challenged i.p., mice deficient in either MPO or NE were markedly more susceptible to bacterial infection and death. In vitro studies suggested that MPO impairs the morphology of bacteria in a distinctive way. Of importance, our in vitro studies found that MPO mediated oxidative inactivation of NE, an enzyme that has been widely implicated in the pathogenesis of various tissue-destructive diseases. This pathway of oxidative inactivation may be physiologically relevant, because activated neutrophils isolated from MPO-deficient mice exhibited increased elastase activity. Our observations provide strong evidence that MPO, like NE, is a key player in the killing of K. pneumoniae bacteria. They also suggest that MPO may modulate NE to protect the host from the tissue-degrading activity of this proteinase.

p-Hydroxyphenylacetaldehyde, the Major Product of l-Tyrosine Oxidation by the Myeloperoxidase-H2O2-Chloride System of Phagocytes, Covalently Modifies ε-Amino Groups of Protein Lysine Residues

Activated human phagocytes employ the myeloperoxidase-H2O2-Cl−system to convert l-tyrosine top-hydroxyphenylacetaldehyde (pHA). We have explored the possibility that pHA covalently reacts with proteins to form Schiff base adducts, which may play a role in modifying targets at sites of inflammation. Because Schiff bases are labile to acid hydrolysis, prior to analysis the adducts were rendered stable by reduction with NaCNBH3. Purified pHA reacted withN α-acetyllysine, an analog of protein lysine residues. The reduced reaction product was identified asN α-acetyl-N ε-(2-(p-hydroxyphenyl)ethyl)lysine by 1H NMR spectroscopy and mass spectrometry. The compoundN ε-(2-(p-hydroxyphenyl)ethyl)lysine (pHA-lysine) was likewise identified in acid hydrolysates of bovine serum albumin (BSA) that were first exposed to myeloperoxidase, H2O2, l-tyrosine, and Cl− and then reduced with NaCNBH3. Other halides (F−, Br−, I−) and the pseudohalide SCN− could not replace Cl− as a substrate in the myeloperoxidase-H2O2-l-tyrosine system. In the absence of the enzymatic system, pHA-lysine was detected in reduced reaction mixtures of BSA, l-tyrosine, and reagent HOCl. In contrast, pHA-lysine was undetectable when BSA was incubated with l-tyrosine and HOBr, peroxynitrite, hydroxyl radical, or a variety of other peroxidases, indicating that the aldehyde-protein adduct was selectively produced by HOCl. Human neutrophils activated in the presence of tyrosine also modified BSA lysine residues. pHA-lysine formation required l-tyrosine and cell activation; it was inhibited by peroxidase inhibitors and catalase, implicating myeloperoxidase and H2O2in the reaction pathway. pHA-lysine was detected in inflamed human tissues that were reduced, hydrolyzed, and then analyzed by mass spectrometry, indicating that the reaction of pHA with proteins may be of physiological importance. These observations raise the possibility that the identification of pHA-lysine in tissues will pinpoint targets where phagocytes inflict oxidative damage in vivo.

Electron Paramagnetic Resonance Detection of Free Tyrosyl Radical Generated by Myeloperoxidase, Lactoperoxidase, and Horseradish Peroxidase*

Phagocytes secrete the heme protein myeloperoxidase, which is present and active in human atherosclerotic tissue. These cells also generate hydrogen peroxide (H2O2), thereby allowing myeloperoxidase to generate a range of oxidizing intermediates and stable end products. When this system acts on l-tyrosine in vitro, it forms o,o′-dityrosine, which is enriched in atherosclerotic lesions. Myeloperoxidase, therefore, may oxidize artery wall proteins in vivo, cross-linking theirl-tyrosine residues. In these studies, we used electron paramagnetic resonance (EPR) spectroscopy to identify an oxidizing intermediate in this reaction pathway and in parallel reactions catalyzed by horseradish peroxidase and lactoperoxidase. Using an EPR flow system to rapidly mix and examine solutions containing horseradish peroxidase, H2O2, and l-tyrosine, we detected free tyrosyl radical (a 2,6 H = 6.3 G,a 3,5 H = 1.6 G, anda β H = 15.0 G). We then used spin trapping techniques with 2-methyl-2-nitrosopropane (MNP) to further identify this intermediate. The resulting three-line spectrum (aN = 15.6 G) was consistent with an MNP/tyrosyl radical spin adduct. Additional MNP spin trapping studies with ring-labeledl-[13C6]tyrosine yielded a characteristic eight-line EPR spectrum (a N = 15.6 G, a 13 C (2) = 8.0 G, a 13 C (1) = 7.1 G, a 13 C (1) = 1.3 G), indicating that the MNP adduct resulted from trapping a carbon-centered radical located on the aromatic ring ofl-tyrosine. This same eight-line spectrum was observed when human myeloperoxidase or bovine lactoperoxidase was substituted for horseradish peroxidase. Furthermore, a partially immobilized MNP/tyrosyl radical spin adduct was detected when we exposed a synthetic polypeptide composed of glutamate and l-tyrosine residues to the myeloperoxidase-H2O2-l-tyrosine system. The broadened EPR signal resulting from this MNP/polypeptide adduct was greatly narrowed by proteolytic digestion with Pronase, confirming that the initial spin-trapped radical was protein-bound. Collectively, these results indicate that peroxidases use H2O2 to convert l-tyrosine to free tyrosyl radical. They also support the idea that free tyrosyl radical initiates cross-linking of l-tyrosine residues in proteins. We suggest that this pathway may play an important role in protein and lipid oxidation at sites of inflammation and in atherosclerotic lesions.

Modification of proteins and lipids by myeloperoxidase

Publisher Summary This chapter describes methods for identifying and quantifying oxidative damage to proteins, lipids, and low density lipoprotein (LDL) that has resulted from myeloperoxidase activity. It discusses general procedures for working with hypochlorous acid 1 (HOC1) and other reactive halogenating agents. The identification of protein and lipid oxidation products that are specifically generated by the myeloperoxidase system, together with the development of methods to detect low levels of these markers in tissues, enables one to probe the role of activated phagocytes in the many diseases associated with oxidative damage. The specificity, sensitivity, and reproducibility of mass spectrometry offer a remarkably powerful technique for this purpose. Identifying additional oxidation products, and developing further mass spectrometric methods for detecting them in vivo , should elicit important insights about the involvement of phagocytes in a variety of human diseases. The chapter also describes methods for isolating LDL from plasma and LDL-like particles from human atherosclerotic tissue and methods for synthesizing, detecting, and quantifying p-hydroxyphenylacetaldehyde (pHA)-lysine and chlorinated sterols, lipid oxidation products generated by myeloperoxidase.

Elevated levels of protein-bound p-hydroxyphenylacetaldehyde, an amino-acid-derived aldehyde generated by myeloperoxidase, are present in human fatty streaks, intermediate lesions and advanced atherosclerotic lesions

Reactive aldehydes might have a pivotal role in the pathogenesis of atherosclerosis by covalently modifying low-density lipoprotein (LDL). However, the identities of the aldehyde adducts that form on LDL in vivo are not yet clearly established. We previously demonstrated that the haem protein myeloperoxidase oxidizes proteins in the human artery wall. We also have shown that p-hydroxyphenylacetaldehyde (pHA), the aldehyde that forms when myeloperoxidase oxidizes L-tyrosine, covalently modifies the N(epsilon)-lysine residues of proteins. The resulting Schiff base can be quantified as N(epsilon)-[2-(p-hydroxyphenyl)ethyl]lysine (pHA-lysine) after reduction with NaCNBH(3). Here we demonstrate that pHA-lysine is a marker for LDL that has been modified by myeloperoxidase, and that water-soluble, but not lipid-soluble, antioxidants inhibit the modification of LDL protein. To determine whether myeloperoxidase-generated aldehydes might modify LDL in vivo, we used a combination of isotope-dilution GC-MS to quantify pHA-lysine in aortic tissues at various stages of lesion evolution. We also analysed LDL isolated from atherosclerotic aortic tissue. Comparison of normal and atherosclerotic aortic tissue demonstrated a significant elevation (more than 10-fold) of the reduced Schiff base adduct in fatty streaks, intermediate lesions and advanced lesions compared with normal aortic tissue. Moreover, the level of pHA-lysine in LDL recovered from atherosclerotic aortic intima was 200-fold that in plasma LDL of healthy donors. These results indicate that pHA-lysine, a specific covalent modification of LDL, is generated in human atherosclerotic vascular tissue. They also raise the possibility that reactive aldehydes generated by myeloperoxidase have a role in converting LDL into an atherogenic lipoprotein.