Increased Superoxide Release Research Articles

Immunoregulatory Roles of IL-10 in Innate Immunity: IL-10 Inhibits Macrophage Production of IFN-γ-Inducing Factors but Enhances NK Cell Production of IFN-γ

AbstractIn our study of the immunoregulatory roles of IL-10 in innate immunity, nonantigenic phagocytosable chitin particles were administered i.v. to IL-10-deficient (knockout (KO)) mice or KO mice pretreated with anti-NK1.1 or anti-IFN-γ Abs. The results established that chitin treatment of KO mice increased superoxide anion release from alveolar macrophages (Mφ) to a level much higher than that in wild-type (WT) mice. The results also suggested that the NK cell is the source of IFN-γ that is primarily responsible for this alveolar Mφ priming. To further study the roles of IL-10-inhibiting chitin-induced IFN-γ production, we used spleen cell cultures. The experiments showed that IL-12, IL-18, and TNF-α, which were produced by chitin-stimulated Mφ, contributed to the IFN-γ-inducing activity of chitin. Our results established that exogenous IL-10 inhibited chitin-induced IFN-γ production in spleen cell cultures from both KO and WT mice. Exogenous IL-10 also inhibited IL-12 and TNF-α production by chitin-stimulated Mφ. Exogenous IL-10 decreased IL-12- or IL-18-induced IFN-γ levels in KO but not in WT NK cell cultures. However, exogenous IL-10 enhanced IFN-γ levels when NK cells were stimulated simultaneously with both IL-12 and IL-18 in KO and WT cultures. Our in vitro data indicate that IL-10 has differential effects on chitin-induced IFN-γ production. However, the inhibitory effects of endogenous IL-10 appear to be dominant in the chitin-induced alveolar Mφ priming response in vivo.

Effects of proteinosis surfactant proteins on the viability of rat alveolar macrophages.

Pulmonary alveolar proteinosis (PAP) is a disease of unknown etiology that is characterized by the accumulation of protein- and lipid-rich insoluble material in alveoli and terminal bronchioles of the lung. Alveolar macrophages (AM) in PAP are reportedly extremely large and have low viability. We investigated substances in lavaged lung material from patients with PAP that induced these cellular changes in rat AM. Rat AM were incubated for various periods with liposomes prepared from lipids and isolated hydrophobic surfactant apoproteins, and cell size, viability, and lactate dehydrogenase release were determined. Of the hydrophobic apoproteins, a dimeric form of surfactant-associated protein-C ([SP-C]2) had the most marked effects. In addition, [SP-C]2 induced increased superoxide anion release at an early phase (6 to 12 h) and an increase in glutathione content at 24 h of incubation. At 3 d after incubation, cellular glutathione and adenosine triphosphate (ATP) content were significantly decreased in cells treated with [SP-C]2, [SP-C]2 was presumed to cause early cell death through increased formation of superoxide anion and the subsequent derangement of cellular metabolism. [SP-C]2 was not removed from cells, and SP-B and SP-C were removed at slower rates than lipids. The changes in macrophages induced by [SP-C]2 may contribute to establishing PAP.

IMMUNOLOGIC AND HAEMATOLOGIC CONSEQUENCES OF CHORIOAMNIONITIS 38

Maternal-fetal interactions involve various hematological and immunological abnormalities, some of which have only recently been clarified. Although maternal infections and chorioamnionitis are known for some time, the recent demonstration of the role of placental infections in maternal and fetal cytokine production and on activation of labor sheds new light on the origin of certain diseases of the newborn infant. Indeed, chorioamnionitis is followed by elevated proinflammatory cytokine levels in the mother and newborn and may be responsible for premature delivery and neonatal asphyxia. Investigations into feto-maternal interactions related to cytokine release have demonstrated that interleukins are intimately connected with the onset of labor and the activation of superoxide anion release by professional phagocytes. Increased inflammatory mediators in the fetus and newborn can cause tissue damage and lung inflammatory injury, and play a key role in the development of RDS and chronic lung disease. In our experience, increased IL-1 beta, IL-6 and IFN-gamma can be found in the mother during labor and increased IL-6 in the newborn during the first hours of life. This increase has been associated with increased superoxide release by WBC of mothers and newborns. Our studies on the the relations between subclinical chorioamnionitis and neonatal outcome have revealed histological chorioamnionitis in 62% of all premature deliveries, and 50% of RDS cases. In particular, in preterm infants, histological chorioamnionitis has been associated with fetal tachycardia, metabolic acidosis, and immune acute phase response at birth. Histological chorioamnionitis may also be responsible for a sharp increase in fetal erythropoietin production leading for an increase in nucleated red blood cells(NRBC) in the fetus and newborn. In 251 newborns with gestational ages of 24-41 weeks, we found a significant correlation between the number of NRBC and the other markers of intrauterine asphyxia and brain damage. In conclusion, maternal infections and placental abnormalities may be followed by abnormally elevated inflammatory mediators and increased erythropoiesis.

Neutrophil superoxide release and interleukin 8 in acute myocardial infarction: distinction between complicated and uncomplicated states.

Superoxide release in neutrophils and sera levels of interleukin 8 (IL-8) were determined in 15 patients with complicated acute myocardial infarction (MI) and 15 patients with uncomplicated MI. All patients showed increased superoxide release in unstimulated and stimulated neutrophils compared with healthy control subjects, indicating priming of these cells. Superoxide release of unstimulated or stimulated neutrophils was found to be significantly higher in patients with complicated MI than in patients with uncomplicated MI. Thrombolytic therapy did not affect the rates of superoxide release. The neutrophil chemoattractant/activator IL-8 was detected in the sera of all patients, with significantly higher levels in those with complicated MI. The highest levels of IL-8 were detected at admission to the Coronary Care Unit and significantly decreased thereafter, suggesting its contribution to neutrophil-mediated tissue injury. The high levels of IL-8 may be one of the major contributors to the priming of neutrophils in these patients.

Effect of Coating with Lung Lining Fluid on the Ability of Fibres to Produce a Respiratory Burst in Rat Alveolar Macrophages

The aim of the study was to develop a simple short-term in vitro assay which would allow us to predict the pathogenicity of fibres based on data already available from in vivo studies. Fibres were used naked (uncoated) or coated with rat IgG, or rat or sheep surfactant. The fibres were used to stimulate superoxide anion release by rat alveolar macrophages. Binding of fibres to rat alveolar macrophages was assessed by optical microscopy. Fibres used in the naked state produced little or no stimulation of superoxide anion from rat alveolar macrophages. When fibres were coated with rat IgG there was a significant increase in superoxide release for all fibre types with the exception of RCF4 and Code 100/475. When fibres were coated with rat or sheep surfactant, there was suppression of the respiratory burst for all fibre types. The observed suppression was not due to a scavenging effect by the surfactant itself, because xanthine/xanthine oxidase generated superoxide was unaffected by surfactant. The suppressive effect was shown to act directly on the macrophages. Comparing naked and coated fibres for their ability to bind to macrophages, it was shown that in general more coated fibres were bound and that increased binding was associated with suppressed superoxide release for both types of surfactant-coated fibres. It was concluded that the nature of the fibre coating is the main factor influencing the interaction between fibres and macrophages. The type of binding through different receptors may either stimulate or switch off the respiratory burst. The assay used here does not, however, allow any predictions to be made regarding the pathogenicity of fibres.

Dual function of human IgA antibodies: inhibition of phagocytosis in circulating neutrophils and enhancement of responses in IL-8-stimulated cells

We have sought to elucidate the responses of human peripheral blood neutrophils to antigenic surfaces complexed with human specific IgA antibodies obtained either as myeloma proteins that recognize staphylococcal alpha-toxin, or from the serum of patients with subacute bacterial endocarditis due to Streptococcus mutans, or from colostrum. In contrast to IgG, IgA antibodies bound to antigen-coated fluorescent microspheres, and subsequently exposed to complement (or not), did not promote phagocytosis, as measured by flow cytometric enumeration of cell-associated microspheres. Instead, IgA antibodies interfered with complement-dependent phagocytosis mediated by IgG antibodies. These properties were shown by different forms of IgA antibodies, including serum and secretory IgA, as well as by monoclonal or polyclonal antibodies. Neutrophils did not respond to the production of superoxide to IgA antibodies complexed with antigen-coated microspheres or with antigen deposited on a solid surface and IgA antibodies suppressed IgG antibody- and complement-mediated superoxide release. However, neutrophils pretreated with interleukin-8 ingested IgA-opsonized microspheres and released superoxide when exposed to IgA antibody-antigen complexes. IgG antibody-antigen complexes did not stimulate increased superoxide release in interleukin-8-treated neutrophils. These findings were consistent with a selective increase in the surface expression of Fc alpha R by interleukin-8-treated neutrophils. We conclude that IgA antibodies interfere with the phagocytic activities of normal circulating human neutrophils and may promote these activities in inflammatory neutrophils activated by interleukin-8 in which Fc alpha R is up-regulated.

Stimulation of human monocytes by anti-CD3 monoclonal antibody: induction of inflammatory mediator release via immobilization of Fc receptor by adsorbed immunoglobulin and T-lymphocytes.

Human monocytes released superoxide anion, prostaglandin E2, leukotriene B4, IL-1, and TNF when exposed to plastic surfaces coated with murine anti-CD3 monoclonal antibody, OKT 3. Stimulation of mediator release by OKT 3 was dependent on the amount of antibody immobilized onto wells of plastic tissue culture plates. Soluble antibody or antibody adsorbed to monocytes and reacted with an aggregating ("cross-linking") second antibody failed to induce mediator release. Monocytes "armed" with OKT 3 formed rosettes with T cells in a fashion indistinguishable from that seen between monocytes and T cells sensitized with OKT 3. Monocytes with adsorbed OKT 3 antibodies released IL-1 beta and TNF-alpha when exposed to unsensitized T cells, although increased superoxide release could not be detected. OKT 4a, a murine IgG2a antibody that reacts with a different T cell epitope (CD4), failed to induce cytokine release from monocytes when cross-linked by T cells or a CD4+ T cell line, even in the presence of IL-2 or IFN-gamma. These data indicate that certain antibodies bound to Fc receptors (FcR) of monocytes may trigger monocyte function when reacting with cells bearing the appropriate target antigens. FcR-mediated signaling resulting in mediator release may be involved in initiating or regulating the immune response. Furthermore, systemically administered monoclonal antibodies may induce inflammatory responses and their attendant symptomatologies via their interaction with FcR-bearing inflammatory cells.

Naloxone is an inappropriate antagonist of Met-enkephalin-modulated superoxide anion release

Met-enkephalin (MENK) increased superoxide anion release by human polymorphonuclear cells (PMNs) at a physiological (10 −10 M) concentration and decreased release at a relatively high (10 −8 M) concentration. Surprisingly, naloxone (NAL), used as a specific antagonist in these experiments, displayed immunomodulatory actions of its own, increasing superoxide anion release at 10 −10 and 10 −8 M concentrations. Although both were effective, the dose-response curves were different for NAL and MENK. NAL and MENK also had a combined influence on O 2 − release. The stimulative effect of 10 −10 M MENK could be abolished by 10 −8 M NAL in seven of eight cases. Unexpectedly, the stimulatory effect of 10 −8 and 10 −10 M NAL could be abrogated by MENK in five of eight cases as well. The fact that NAL and MENK mutually interfere with one another in their effect upon O 2 − release by human PMNs discredits NAL as an appropriate antagonist in this system.

Acute ethanol intoxication stimulates superoxide anion production by in situ perfused rat liver.

This study examines the generation of superoxide anion by the perfused rat liver after ethanol intoxication and acute endotoxemia to assess the potential importance of oxygen-derived free radicals in the ethanol-induced hepatic pathological condition. Hepatic superoxide anion production of 0.65 +/- 0.06 nmol/min/gm liver weight was measured 1 hr after ethanol infusion; it reached a peak value of 0.8 +/- 0.07 at 3 hr and was reduced to 0.11 +/- 0.01 by 7 hr. In a group of animals, 4-methylpyrazole was injected 5 min before the administration of ethanol to determine whether the metabolism of ethanol moiety is necessary for the observed effects. However, no significant inhibition of superoxide production was observed after 4-methylpyrazole administration. Introduction of ibuprofen into the perfused liver abolished superoxide anion production, suggesting that arachidonic acid metabolites may play an important role in superoxide generation under these conditions. Endotoxin, a potent activator of macrophages, has also been associated with increased superoxide release by the liver. Therefore the combined impact of ethanol and endotoxin on superoxide production by the liver was also examined. Acute ethanol intoxication inhibited the endotoxin-mediated superoxide anion generation by the perfused liver. These data indicate that the ethanol-mediated superoxide production and the ethanol-induced inhibition of the lipopolysaccharide-enhanced free-radical generation by the liver may have a pathophysiological significance in tissue injury and in resistance to infection.

Increased superoxide anion release by peritoneal macrophages in mice with a chronic infection of lactic dehydrogenase virus

The function of macrophages in mice chronically infected by lactic dehydrogenase virus (LDV) was studied. Superoxide anion ( O 2 −) release was examined by using peritoneal macrophages. O 2 − release increaed markedly from 3 weeks to 12 months, but not at 1 week post infection. O 2 − release was 1·2 to 1·5 times greater than in uninfected mice. Increased O 2 − release from macrophages in LDV-infected mice may explain, at least in part, suppressive effects on tumour growth seen in the chronic phase of infection.

Modulation of transmembrane signalling in HL-60 granulocytes by tumour necrosis factor-alpha.

Differentiated HL-60 granulocytes were used to study the mechanism by which tumour necrosis factor-alpha (TNF) enhances responses to N-formyl-methionyl-leucylphenylalanine (FMLP). Cultivation of differentiated HL-60 cells with 100 units of TNF/ml for 24 h resulted in a 3-fold increase in superoxide release and 4-fold increase in prostaglandin E2 production on stimulation with 1 microM-FMLP. On the other hand, cultivation with TNF failed to increase phorbol diester stimulation of superoxide release. Formyl-peptide-receptor expression determined on isolated membranes from cells cultivated with TNF (TNF-M) was increased by 50% compared with membranes from control cells (NM). Similarly, FMLP binding to intact HL-60 cells was increased by cultivation with TNF. Guanine-nucleotide-binding proteins (G-protein) levels were not different between TNF-M and NM, as determined by pertussis-toxin-catalysed ADP-ribosylation and by immunoblotting with antisera recognizing alpha i2 subunit. Binding of guanosine 5'-[gamma-thio]triphosphate and GTP hydrolysis stimulated by FMLP were enhanced by about 50% in TNF-M. The efficiency of G-protein activation by formyl-peptide receptors did not differ between TNF-M and NM. TNF regulates expression of formyl-peptide receptors independently of G-protein levels. The regulation of receptor expression is one mechanism by which TNF enhances cell responses to formylated peptides.

Superoxide anion release from blood and bone marrow neutrophils is altered by endotoxemia.

In vivo endotoxin infusion produces neutrophil-mediated acute lung injury and increases superoxide anion release from phorbol myristate acetate (PMA)-stimulated blood neutrophils collected 18-24 hours after the infusion. Because the turnover time of circulating blood neutrophils is only 6-8 hours, it was hypothesized that the prolonged increase in superoxide anion release from peripheral blood neutrophils is associated with increased superoxide anion release from bone marrow neutrophils. To test this hypothesis, two doses of Escherichia coli endotoxin (5.0 and 0.5 micrograms/kg) were infused into chronically instrumented awake sheep. Blood and bone marrow neutrophils were collected 24 hours after the infusion, and superoxide anion release from unstimulated and PMA-stimulated neutrophils was measured in vitro. Endotoxin infusion produced an increase in pulmonary microvascular permeability, in intravascular activation (degranulation) of blood neutrophils, and in circulating blood neutrophils 24 hours after the infusion. High-dose endotoxin (5.0 micrograms/kg; n = 4) increased superoxide anion release from unstimulated peripheral blood neutrophils (2.25 +/- 0.38 times baseline [p less than or equal to 0.05]) and from peripheral blood neutrophils stimulated with 10(-9) M PMA in vitro (1.46 +/- 0.55 times baseline). Low-dose endotoxin (0.5 micrograms/kg; n = 5), on the other hand, did not alter superoxide anion release from peripheral blood neutrophils. Bone marrow neutrophils could not be isolated reproducibly after high-dose endotoxin because of leukoaggregation. Bone marrow neutrophils were isolated after low-dose endotoxin infusion. Stimulation of these cells with 10(-9) M PMA in vitro resulted in a two- to fourfold increase above control release (p less than or equal to 0.05). Increased superoxide anion release from both peripheral blood and bone marrow neutrophils occurred in the absence of circulating endotoxin, as measured by a Limulus assay. These results show that the prolonged increase in superoxide anion release from peripheral blood neutrophils is associated with an increase in the superoxide anion release from bone marrow neutrophils. Furthermore, the recruitment of affected bone marrow neutrophils into peripheral blood may explain the increased superoxide anion release from blood neutrophils 24 hours after endotoxin infusion.

Mechanisms of pulmonary edema induced by a diacylglycerol second messenger

We investigated the effect of dioctanoylglycerol (DOG), a second messenger of protein kinase C (PKC) activation, in the absence and presence of neutrophils in isolated perfused guinea pig lung. DOG was given after a base-line isogravimetric steady-state period. Pulmonary capillary pressure (Ppc) and change in lung weight (delta W) were monitored at 15, 30, and 60 min. Capillary filtration coefficient (Kf,c, an index of vascular permeability) was measured during base-line period and at 30 min. DOG increased the Ppc and delta W at 30 and 60 min, and the Kfc at 30 min. Monooctanoylglycerol, a monoacylglycerol that does not activate PKC, had no effect on Ppc, Kf,c, and delta W. Pretreatment with two different PKC inhibitors, 1-(5-isoquinolinylsulfonyl)-2-methyl piperazine or staurosporin, prevented the pulmonary response to DOG. With neutrophils present, DOG caused greater increases in delta W and the (wet-dry)-to-dry wt ratio compared with DOG group. Response to DOG+ neutrophils was due to oxygen radical production because it was prevented by pretreatment with catalase and because DOG increased superoxide release from neutrophils. PKC activation using DOG in the isolated lung results in pulmonary edema mediated by increases in capillary pressure and vascular permeability. Lung weight-gain response to DOG is greater in the presence of neutrophils. Response to DOG+ neutrophils is mediated by oxygen radicals.

Phagocytosis induction of chemiluminescence and chemoattractant increased superoxide anion release from activated human alveolar macrophages in asthma

Human alveolar macrophages (AM) were demonstrated to generate reactive toxic derivatives of oxygen in many pulmonary disorders. These cells are involved in local inflammation which characterizes bronchial asthma. In the present work, we studied the ability of stimulated macrophages from healthy volunteers, and asthmatic patients to generate oxygen species in vitro. AM obtained by bronchoalveolar lavage were purified by adherence. The production of oxygen species was measured by luminol-enhanced chemiluminescence (CL) after challenge with opsonized zymosan. The maximal values were significantly (p less than 0.03 and p less than 0.01) higher in AM from asthmatics than in AM from healthy subjects. A significant correlation (p less than 0.01) was observed between maximal value of CL and the severity of asthma as assessed by the clinical score. But, no difference was observed between AM from asthmatics in a stable state and healthy subjects. On the other hand, assays for superoxide anion generation emphasized the activation state of these macrophages stimulated by formyl-peptides.

Volume dependent polymorphonuclear leukocytes fractions isolated by counterflow centrifugal elutriation: PMN volume does not correlate with PMN age.

We have previously reported that human peripheral blood polymorphonuclear leukocytes can be separated into at least six volume dependent fractions using counterflow centrifugal elutriation (CCE). Larger volume was shown to correlate with increased superoxide release with either the chemotactic peptide F-met-leu-phe (FMLP) or the tumor promotor phorbol myristate acetate (PMA). To help explain these findings we have examined volume dependent PMN fractions (VDPF) for PMN functions felt to be correlated to PMN age, ie, alkaline phosphatase activity, phagocytosis of opsonized particles, adherence to plastic, and directed movement to FMLP and zymosan activated serum. PMN alkaline phosphatase activity was found to be low in the smallest PMNs, increasing in the PMNs of intermediate size, and then decreasing again in the largest PMN functions. A similar relationship was noted for PMN phagocytic activity. No significant difference among VDPF in adherence to plastic or chemotaxis was found. Furthermore, no difference among VDPF was seen for receptor number or binding affinity of the ligands FMLP or phorbol dibutyrate. Total cellular activity of the NADPH dependent oxidase was, however, 200% greater in the largest compared to the smallest VDPF. Because of the lack of consistent correlation between "age" related PMN function and PMN volume, it is unlikely that PMN volume represents a manifestation of PMN age. It is likely that the increased oxidase activity seen among larger VDPF accounts for the more rapid oxidative burst previously noted.

Increased superoxide anion release from human endothelial cells in response to cytokines.

To study the effects of macrophage and lymphocyte-derived factors on superoxide anion (O2-) generation and release from human umbilical vein endothelial cells (EC), cultured EC were stimulated by ultrapure interleukin 1 (IL 1) and recombinant interferon-gamma (IFN-gamma), and the O2- released into the supernatant was measured. Both of these cytokines enhanced O2- release in a dose and time-dependent manner. Addition of a combination of IL 1 and IFN-gamma, each in submaximal concentration, produced an additive effect on O2- release. It would appear from these findings that cytokines released by macrophages and lymphocytes during inflammatory reactions can promote O2- generation and release from human EC. O2- released from EC may alter the basement membrane of blood vessels and the surrounding connective tissue, and in this way promote the vascular injury and angiogenesis associated with local inflammation.

Role of macrophage lipids in regulating tumoricidal activity

Peritoneal macrophages (Mφ) from mice became cytotoxic after incubation with lymphokine (LK); tumoricidal activity was evident with Mφ treated with LK for 4 hr, became maximal after 8–12 hr of incubation, and decreased to control levels by 24–36 hr. LK induced marked changes in Mφ lipid composition: cellular content of cholesterol (CHOL) and polyunsaturated fatty acid (UFA) content of cellular lipids (especially 18:3) increased two- to threefold after 8 hr when the cells showed maximal tumoricidal activity. Cellular lipid and fatty-acid content returned to control levels by 24 hr when the Mφ had lost tumoricidal activity. These changes were not observed with equal numbers of Mφ cultured in control supernatants. To analyze the role of CHOL and UFA in Mφ tumor cytotoxicity, casein-induced peritoneal Mφ were enriched in CHOL or linolenic acid (18:3) and then tested for their ability to kill 1023 tumor cells. The 18:3-enriched cells were markedly tumoricidal, whereas controls cultured in delipidized medium alone or enriched with saturated fatty acid (18:0) were not cytotoxic. CHOL-enriched Mφ were not tumoricidal; indeed, these cells were inhibited in their killing after treatment with LK compared to Mφ cultured in delipidized medium with LK alone. The effects of 18:3 and CHOL enrichment of the Mφ on their metabolic status, inflammatory function, and tumor cell-binding capacity were tested. The 18:3-enriched Mφ were depressed in their ability to synthesize protein and in phagocytic activity compared to controls; these cells showed a transient increase in superoxide release. Mφ cultured with 18:3 for 48 hr were also cytotoxic for P815 tumor cells, but did not show an enhanced capacity for P815 binding compared to controls. CHOL-enriched Mφ were similar to control cells in their protein synthesizing and phagocytic activities; these cells also showed an early transient increase in superoxide release. CHOL-enriched Mφ were not cytotoxic for P815 cells, but bound the tumor cells more readily than did the 18:3-enriched Mφ. The data suggest that endogenous levels of 18:3 and CHOL can regulate Mφ tumor cytotoxicity, but not through regulation of Mφ protein synthesis, oxidative metabolism, or augmented capacity for tumor target binding.

Oxidative Metabolism of Alveolar Macrophages from Young Asymptomatic Cigarette Smokers: Increased Superoxide Anion Release and Its Potential Consequences

Alveolar macrophages (AM) have been implicated in the pathogenesis of the centrilobular emphysema related to cigarette smoking. Clusters of AM lodge in the bronchioles of smokers before the development of emphysema, and persist as tissue destruction and fibrosis progress. 1 Niewoehner DE Kleineman J Rice DB Pathologic changes in the peripheral airways of young cigarette smokers. N Engl J Med. 1974; 294: 755 Crossref Scopus (681) Google Scholar , 2 Cosio M Ghezzo H Ghezzo JC et al. The relations between structural changes in small airways and pulmonary function tests. N Engl J Med. 1978; 298: 1277 Crossref PubMed Scopus (589) Google Scholar While these observations support a role for AM in the induction of emphysema, the mechanism by which AM may cause emphysema is not known.

Oxidative Metabolism of Alveolar Macrophages from Young Asymptomatic Cigarette Smokers: Increased Superoxide Anion Release and Its Potential Consequences

Oxidative Metabolism of Alveolar Macrophages from Young Asymptomatic Cigarette Smokers: Increased Superoxide Anion Release and Its Potential Consequences