Michael L. McCormick

Ghrelin Inhibits Proinflammatory Responses and Nuclear Factor-κB Activation in Human Endothelial Cells

Background—Ghrelin is a novel growth hormone–releasing peptide that has been shown to improve cachexia in heart failure and cancer and to ameliorate the hemodynamic and metabolic disturbances in septic shock. Because cytokine-induced inflammation is critical in these pathological states and because the growth hormone secretagogue receptor has been identified in blood vessels, we examined whether ghrelin inhibits proinflammatory responses in human endothelial cells in vitro and after administration of endotoxin to rats in vivo. Methods and Results—Human umbilical vein endothelial cells (HUVECs) were treated with or without tumor necrosis factor-&agr;(TNF-&agr;), and induction of proinflammatory cytokines and mononuclear cell adhesion were determined. Ghrelin (0.1 to 1000 ng/mL) inhibited both basal and TNF-&agr;–induced cytokine release and mononuclear cell binding. Intravenous administration of ghrelin also inhibited endotoxin-induced proinflammatory cytokine production in rats in vivo. Ghrelin inhibited H2O2-induced cytokine release in HUVECs, suggesting that the peptide blocks redox-mediated cellular signaling. Moreover, ghrelin inhibited basal and TNF-&agr;–induced activation of nuclear factor-κB. Des-acyl ghrelin had no effect on TNF-&agr;–induced cytokine production in HUVECs, suggesting that the antiinflammatory effects of ghrelin require interaction with endothelial growth hormone secretagogue receptors. Conclusions—Ghrelin inhibits proinflammatory cytokine production, mononuclear cell binding, and nuclear factor-κB activation in human endothelial cells in vitro and endotoxin-induced cytokine production in vivo. These novel antiinflammatory actions of ghrelin suggest that the peptide could play a modulatory role in atherosclerosis, especially in obese patients, in whom ghrelin levels are reduced.

Oxidative Responses of Human and Murine Macrophages During Phagocytosis of Leishmania chagasi

Leishmania chagasi, the cause of South American visceral leishmaniasis, must survive antimicrobial responses of host macrophages to establish infection. Macrophage oxidative responses have been shown to diminish in the presence of intracellular leishmania. However, using electron spin resonance we demonstrated that murine and human macrophages produce O2− during phagocytosis of opsonized promastigotes. Addition of the O2− scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl to cultures resulted in increased infection, suggesting that O2− enhances macrophage leishmanicidal activity. The importance of NO· produced by inducible NO synthase (iNOS) in controlling murine leishmaniasis is established, but its role in human macrophages has been debated. We detected NO· in supernatants from murine, but not human, macrophages infected with L. chagasi. Nonetheless, the iNOS inhibitor NG-monomethyl-l-arginine inhibited IFN-γ-mediated intracellular killing by both murine and human macrophages. According to RNase protection assay and immunohistochemistry, iNOS mRNA and protein were expressed at higher levels in bone marrow of patients with visceral leishmaniasis than in controls. The iNOS protein also increased upon infection of human macrophages with L. chagasi promastigotes in vitro in the presence of IFN-γ. These data suggest that O2− and NO· each contribute to intracellular killing of L. chagasi in human and murine macrophages.

Role of Oxidative Stress in the Pathogenesis of Abdominal Aortic Aneurysms

The role of inflammation in the pathogenesis of abdominal aortic aneurysms (AAA) is well established. The inflammatory process leads to protease-mediated degradation of the extracellular matrix and apoptosis of smooth muscle cells (SMC), which are the predominant matrix synthesizing cells of the vascular wall. These processes act in concert to progressively weaken the aortic wall, resulting in dilatation and aneurysm formation. Oxidative stress is invariably increased in, and contributes importantly to, the pathophysiology of inflammation. Moreover, reactive oxygen species (ROS) play a key role in regulation of matrix metalloproteinases and induction of SMC apoptosis. ROS may also contribute to the pathogenesis of hypertension, a risk factor for AAA. Emerging evidence suggests that ROS and reactive nitrogen species (RNS) are associated with AAA formation in animal models and in humans. Although experimental data are limited, several studies suggest that modulation of ROS production or activity may suppress AAA formation and improve experimental outcome in rodent models. Although a number of enzymes can produce injurious ROS in the vasculature, increasing evidence points toward a role for NADPH oxidase as a source of oxidative stress in the pathogenesis of AAA.

Deletion of p47phox Attenuates Angiotensin II–Induced Abdominal Aortic Aneurysm Formation in Apolipoprotein E–Deficient Mice

Background— Angiotensin II (Ang II) contributes to vascular pathology in part by stimulating NADPH oxidase activity, leading to increased formation of superoxide (O2−). We reported that O2− levels, NADPH oxidase activity, and expression of the p47phox subunit of NADPH oxidase are increased in human abdominal aortic aneurysms (AAAs). Here, we tested the hypothesis that deletion of p47phox will attenuate oxidative stress and AAA formation in Ang II–infused apoE−/− mice. Methods and Results— Male apoE−/− and apoE−/−p47phox−/− mice received saline or Ang II (1000 ng · kg−1 · min−1) infusion for 28 days, after which abdominal aortic weight and maximal diameter were determined. Aortic tissues and blood were examined for parameters of aneurysmal disease and oxidative stress. Ang II infusion induced AAAs in 90% of apoE−/− versus 16% of apo−/−p47phox−/− mice (P<0.05). Abdominal aortic weight (14.1±3.2 versus 35.6±9.0 mg), maximal aortic diameter (1.5±0.2 versus 2.4±0.4 mm), aortic NADPH oxidase activity, and parameters of oxidative stress were reduced in apoE−/−p47phox−/− mice compared with apoE−/− mice (P<0.05). In addition, aortic macrophage infiltration and matrix metalloproteinase-2 activity were reduced in apoE−/−p47phox−/− mice compared with apoE−/− mice. Deletion of p47phox attenuated the pressor response to Ang II; however, coinfusion of phenylephrine with Ang II, which restored the Ang II pressor response, did not alter the protective effects of p47phox deletion on AAA formation. Conclusions— Deletion of p47phox attenuates Ang II–induced AAA formation in apoE−/− mice, suggesting that NADPH oxidase plays a critical role in AAA formation in this model.

Vitamin E Inhibits Abdominal Aortic Aneurysm Formation in Angiotensin II–Infused Apolipoprotein E–Deficient Mice

Background—Abdominal aortic aneurysms (AAAs) in humans are associated with locally increased oxidative stress and activity of NADPH oxidase. We investigated the hypothesis that vitamin E, an antioxidant with documented efficacy in mice, can attenuate AAA formation during angiotensin II (Ang II) infusion in apolipoprotein E–deficient mice. Methods and Results—Six-month-old male apolipoprotein E–deficient mice were infused with Ang II at 1000 ng/kg per minute for 4 weeks via osmotic minipumps while consuming either a regular diet or a diet enriched with vitamin E (2 IU/g of diet). After 4 weeks, abdominal aortic weight and maximal diameter were determined, and aortic tissues were sectioned and examined using biochemical and histological techniques. Vitamin E attenuated formation of AAA, decreasing maximal aortic diameter by 24% and abdominal aortic weight by 34% (P<0.05, respectively). Importantly, animals treated with vitamin E showed a 44% reduction in the combined end point of fatal+nonfatal aortic rupture (P<0.05). Vitamin E also decreased aortic 8-isoprostane content (a marker of oxidative stress) and reduced both aortic macrophage infiltration and osteopontin expression (P<0.05, respectively). Vitamin E treatment had no significant effect on the extent of aortic root atherosclerosis, activation of matrix metalloproteinases 2 or 9, serum lipid profile, or systolic blood pressure. Conclusions—Vitamin E ameliorates AAAs and reduces the combined end point of fatal+nonfatal aortic rupture in this animal model. These findings are consistent with the concept that oxidative stress plays a pivotal role in Ang II–driven AAA formation in hyperlipidemic mice.

Interaction of the Pseudomonas aeruginosa secretory products pyocyanin and pyochelin generates hydroxyl radical and causes synergistic damage to endothelial cells. Implications for Pseudomonas-associated tissue injury.

Pyocyanin, a secretory product of Pseudomonas aeruginosa, has the capacity to undergo redox cycling under aerobic conditions with resulting generation of superoxide and hydrogen peroxide. By using spin trapping techniques in conjunction with electron paramagnetic resonance spectrometry (EPR), superoxide was detected during the aerobic reduction of pyocyanin by NADH or porcine endothelial cells. No evidence of hydroxyl radical formation was detected. Chromium oxalate eliminated the EPR spectrum of the superoxide-derived spin adduct resulting from endothelial cell exposure to pyocyanin, suggesting superoxide formation close to the endothelial cell plasma membrane. We have previously reported that iron bound to the P. aeruginosa siderophore pyochelin (ferripyochelin) catalyzes the formation of hydroxyl free radical from superoxide and hydrogen peroxide via the Haber-Weiss reaction. In the present study, spin trap evidence of hydroxyl radical formation was detected when NADH and pyocyanin were allowed to react in the presence of ferripyochelin. Similarly, endothelial cell exposure to pyocyanin and ferripyochelin also resulted in hydroxyl radical production which appeared to occur in close proximity to the cell surface. As assessed by 51Cr release, endothelial cells which were treated with pyocyanin or ferripyochelin alone demonstrated minimal injury. However, endothelial cell exposure to the combination of pyochelin and pyocyanin resulted in 55% specific 51Cr release. Injury was not observed with the substitution of iron-free pyochelin and was diminished by the presence of catalase or dimethyl thiourea. These data suggest the possibility that the P. aeruginosa secretory products pyocyanin and pyochelin may act synergistically via the generation of hydroxyl radical to damage local tissues at sites of pseudomonas infection.

Production of Brominating Intermediates by Myeloperoxidase A TRANSHALOGENATION PATHWAY FOR GENERATING MUTAGENIC NUCLEOBASES DURING INFLAMMATION

The existence of interhalogen compounds was proposed more than a century ago, but no biological roles have been attributed to these highly oxidizing intermediates. In this study, we determined whether the peroxidases of white blood cells can generate the interhalogen gas bromine chloride (BrCl). Myeloperoxidase, the heme enzyme secreted by activated neutrophils and monocytes, uses H2O2 and Cl− to produce HOCl, a chlorinating intermediate. In contrast, eosinophil peroxidase preferentially converts Br− to HOBr. Remarkably, both myeloperoxidase and eosinophil peroxidase were able to brominate deoxycytidine, a nucleoside, and uracil, a nucleobase, at plasma concentrations of Br− (100 μm) and Cl− (100 mm). The two enzymes used different reaction pathways, however. When HOCl brominated deoxycytidine, the reaction required Br− and was inhibited by taurine. In contrast, bromination by HOBr was independent of Br− and unaffected by taurine. Moreover, taurine inhibited 5-bromodeoxycytidine production by the myeloperoxidase-H2O2-Cl−- Br− system but not by the eosinophil peroxidase-H2O2-Cl−-Br−system, indicating that bromination by myeloperoxidase involves the initial production of HOCl. Both HOCl-Br− and the myeloperoxidase-H2O2-Cl−-Br−system generated a gas that converted cyclohexene into 1-bromo-2-chlorocyclohexane, implicating BrCl in the reaction. Moreover, human neutrophils used myeloperoxidase, H2O2, and Br− to brominate deoxycytidine by a taurine-sensitive pathway, suggesting that transhalogenation reactions may be physiologically relevant. 5-Bromouracil incorporated into nuclear DNA is a well known mutagen. Our observations therefore raise the possibility that transhalogenation reactions initiated by phagocytes provide one pathway for mutagenesis and cytotoxicity at sites of inflammation.

Lactoferrin Binds CpG-Containing Oligonucleotides and Inhibits Their Immunostimulatory Effects on Human B Cells

Unmethylated CpG dinucleotide motifs in bacterial DNA, as well as oligodeoxynucleotides (ODN) containing these motifs, are potent stimuli for many host immunological responses. These CpG motifs may enhance host responses to bacterial infection and are being examined as immune activators for therapeutic applications in cancer, allergy/asthma, and infectious diseases. However, little attention has been given to processes that down-modulate this response. The iron-binding protein lactoferrin is present at mucosal surfaces and at sites of infection. Since lactoferrin is known to bind DNA, we tested the hypothesis that lactoferrin will bind CpG-containing ODN and modulate their biological activity. Physiological concentrations of lactoferrin (regardless of iron content) rapidly bound CpG ODN. The related iron-binding protein transferrin lacked this capacity. ODN binding by lactoferrin did not require the presence of CpG motifs and was calcium independent. The process was inhibited by high salt, and the highly cationic N-terminal sequence of lactoferrin (lactoferricin B) was equivalent to lactoferrin in its ODN-binding ability, suggesting that ODN binding by lactoferrin occurs via charge-charge interaction. Heparin and bacterial LPS, known to bind to the lactoferricin component of lactoferrin, also inhibited ODN binding. Lactoferrin and lactoferricin B, but not transferrin, inhibited CpG ODN stimulation of CD86 expression in the human Ramos B cell line and decreased cellular uptake of ODN, a process required for CpG bioactivity. Lactoferrin binding of CpG-containing ODN may serve to modulate and terminate host response to these potent immunostimulatory molecules at mucosal surfaces and sites of bacterial infection.

Manganese superoxide dismutase in normal and transformed human embryonic lung fibroblasts

The manganese superoxide dismutase (MnSOD) activity of W138 human embryonic lung fibroblasts and SV40-transformed WI38 cells was measured. Under various growth conditions, the transformed cells always had lower MnSOD activity than their normal cell counterparts. The activity of MnSOD changes greatly with the growth conditions in the WI38 cells, while the MnSOD activity in the tumor cells remained more constant. The amount of immunoreactive MnSOD was measured by Western blotting. In all cases studied, the amount of immunoreactive MnSOD was lower in the transformed cells than in the normal cells.

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.