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Subjects with neutrophil myeloperoxidase (MPO) deficiency have been rarely reported. In part this may be due to the lack of a simple screening technique that would detect them. With the routine use of a cytochemical leukocyte differential counter that employs MPO stains, over a 40-mo a period 8 unrelated probands with partial MPO-deficiency and one with complete deficiency were identified. Family studies have identified 23 additional, partially deficient subjects. The largest pedigrees demonstrate that the carrier state or partial MPO deficiency is inherited in an autosomal dominant pattern. Leukocytes from a subject with complete MPO deficiency and from some subjects with partial deficiency have impaired bactericidal activity against S. aureus. Superoxide generation was increased and chemiluminescence decreased in MPO-deficient leukocytes. Eleven subjects were prospectively followed for 18-30 mo. Only two partially deficient subjects have had serious infections consisting of recurrent streptococcal cellulitis and aseptic meningitis. These data suggest that leukocyte MPO deficiency is a common inherited defect that results in minimal clinical problems, supporting the concept of multiple leukocyte bacterial killing systems.

Oxidative metabolic responses of polymorphonuclear leukocytes were assessed in 24 stressed neonates and 22 well, term infants utilizing particulate and soluble stimuli. Stressed neonatal PMNs demonstrated a depressed CL response to zymosan, whereas O2- production for both normal and stressed PMNs of neonates was significantly elevated compared to that in PMNs from adults. These results were not ascribable to phagocytosis since it was comparable in all groups using radiolabeled bacteria. Stressed neonates' PMN responses to soluble stimuli were significantly elevated when compared with those from well neonate and adult controls. Enhanced responses were most prominent in the most severely stressed infants. Treatment of neonates' PMNs with the antioxidants vitamin E and DHB partially corrected the abnormalities, suggesting peroxidative damage to the PMN membrane had occurred. The oxidative metabolic abnormalities of neonates' PMNs are consistent with either a defect in HMPS activity, a defect in functioning of the later portions of the respiratory burst, or a stimulus-specific abnormality in respiratory burst activity.

Neutrophils were preincubated with 17 beta-estradiol and progesterone to determine the effects of these hormones on chemotactic peptide-stimulated superoxide anion (O2-) generation and degranulation. At pharmacologic levels 17 beta-estradiol was more active than progesterone with respect to inhibition of O2- generation as well as degranulation. An increase of preincubation time from 5 minutes to 25 minutes increased the percent inhibition. When 17 beta-estradiol and progesterone were combined at levels which approximate those measured during gestation, there was small but significant inhibition of O2- generation. Dexamethasone at equal molar concentration inhibited O2- generation only after 25 minutes of preincubation and at no time reached the level of inhibition attained by either of the sex hormones alone. Both estradiol and progesterone at pharmacologic levels significantly inhibited beta-glucuronidase and lysozyme release, whereas dexamethasone did not inhibit degranulation despite prolonged preincubation. Neutrophils isolated from women during various phases of the menstrual cycle and during the third trimester of pregnancy did not differ with respect to chemotactic peptide-stimulated O2- generation. These data suggest that inhibition of neutrophil responses requires the continuous presence of pharmacologic levels of estradiol and progesterone.

The study of polymorphonuclear neutrophil (PMN) surface receptor expression provides a means for the assessment of PMN function and state of cellular activation. In this study, we characterized binding of the chemotactic peptide FMLP to whole PMN, with particular attention to those variables that may account for the wide variation reported in the literature. These included avoidance of oxidized FMLP as a radioligand contaminant, determination of the optimal cold ligand concentration necessary for achieving minimal nonspecific binding throughout the range of radioligand concentrations used in saturation experiments (greater than or equal to 5 x 10(-5) M), avoidance of radioligand concentrations that equal or exceed receptor saturation and are not suitable for Scatchard analysis (greater than or equal to 60 to 80 nM), and avoidance of inadvertent receptor mobilization due to room temperature PMN isolation techniques and cell warming. PMN isolated and maintained at 4 degrees C expressed a single, high affinity population of FMLP receptors (approximately 6000 receptors per cell) with a KD of 15.5 nM. These characteristics, and in particular the single-affinity nature of the expressed FMLP receptor site, were derived from saturation experiments and confirmed with agonist competition studies. PMN subjected to room temperature isolation or 37 degrees C warming exhibited a 2.5-fold increase in FMLP receptor expression (approximately 15,000 receptors per cell) without changes in receptor affinity. These latter PMN, in correlation with increased receptor expression, had increased initial, maximal rates of FMLP-induced superoxide generation (10.2 vs 6.3 nmol/min/10(6) PMN for cells isolated and maintained at 4 degrees C) as a manifestation of their functional activation. The avoidance of inadvertent cellular activation during PMN isolation is essential to studies of PMN function, activation and the role of FMLP receptor expression/mobilization in these processes.

Hematoporphyrin derivative and light in the presence of cysteine or glutathione were found to convert oxygen to superoxide and hydrogen peroxide at pH less than approx. 6.5, while at pH greater than 6.5 no superoxide or hydrogen peroxide production was observed. However, at pH values greater than 6.5 the rate of oxygen consumption increased. This rate paralleled the acid dissociation curve of the cysteine thiol group and is consistent with the chemical quenching of 1O2 by cysteine. The superoxide and hydrogen peroxide formation observed below pH 6.5 appeared not to be related to the singlet oxygen production of hematoporphyrin derivative. In addition, superoxide and hydrogen peroxide production was observed with hematoporphyrin derivative and light in the presence of NADH, both above and below pH 6.5. Direct detection of singlet oxygen luminescence at 1268 nm in the hematoporphyrin derivative-light system (2H2O as solvent) revealed an apparent linear increase in the singlet oxygen emission intensity as the p2H was raised from 7.0 to 10.0. Azide efficiently quenched this observed emission. In addition, at p2H 7.4, 1 mM cysteine resulted in a 40% reduction of the singlet oxygen luminescence, while at p2H 9.4 the signal was quenched by over 95% (under the experimental conditions employed). In total, we interpret these results as consistent with the chemical quenching of 1O2 by the ionized thiol group of cysteine.

Previous studies have demonstrated that human eosinophils have a more active hexose monophosphate shunt than neutrophils. However, measurements of superoxide anion production have been variable. In this study human eosinophils are shown to produce one and a half times as much superoxide as neutrophils during a 2-h incubation. The rates of production differed only after the first 30 min of incubation. The production was inhibited by superoxide dismutase in a dose-responsive manner.

In concluding this eclectic review, I believe that we can say with certainty that coronary atherosclerosis has its origins in childhood, at least by age 10 and possibly earlier. Endothelial changes follow lipid accumulation, but, with present methods, there is no evidence that endothelial injury precedes lipid accumulation. Proliferation is a prominent feature of accelerated experimental atherosclerosis, but we do not see evidence that proliferation is a major feature of early naturally occurring human atherosclerosis. The prominence of the monocyte-macrophage in the earliest detectable lesions of atherosclerosis in childhood justifies the current interest in monocyte biology. Hyperlipidemia, in the sense of the average high levels common in our population, seems almost certain to contribute to accelerated atherogenesis in children, but conclusive proof is still lacking. Mild hypertension in children, or even the high range of normal blood pressure, may accelerate the transition of innocuous childhood fatty streaks to more ominous fibrous plaques. The putative relationship of adult coronary heart disease risk factors to the childhood lesions of atherosclerosis is largely based on extrapolation from adult data. Direct evidence bearing on these relationships, however difficult to obtain, would be helpful in designing and promoting effective preventive regimens for children.

Stimulation of granulocyte (PMN) superoxide (O2-) production by concanavalin-A (Con-A) can be monitored continuously in the spectrophotometer. Both the rate of activation and final activity of the O2--generating system is dependent on the concentration of Con-A. Alpha methylmannoside (alpha MM) can prevent Con-A, but not phorbol myristate acetate (PMA) or zymosan, induced O2- production. Alpha MM inhibits both the rate of activation and the final rate of O2- production. When alpha MM is added after the attainment of a maximal rate of O2- production with Con-A, O2- production continues for another minute before it ceases. When PMA is added to such treated cells, it restores O2- production. Although the inhibition of O2- production by alpha MM on previously activated cells requires time, most of the bound concanavalin-A is removed immediately after the addition of alpha MM. Treatment of cells with L-1-tosylamido-2-phenylethyl-chloromethyl ketone (TPCK) prevents activation of PMN by Con-A to a greater extent than it does for either PMA or zymosan. TPCK has no effect on the binding of Con-A. TPCK, when added after Con-A, will inactivate O2- production by the cells. The addition of PMA after TPCK treatment restores O2--generating activity. Membrane-enriched particles from PMN activated with Con-A, alpha MM, and PMA demonstrate that the change in O2- production seen by whole cells is due to an alteration of the activity of the NADPH oxidase. Thus, Con-A stimulation of human PMN O2- production can be prevented and reversed by the addition of either alpha MM or TPCK and that PMA can reactivate Con-A and either alpha MM- or TPCK-treated cells. The activation, inactivation, and reactivation occur as a result of changes in the plasma membrane NADPH-dependent O2--generating enzyme.

The U937 human monocytic cell line was studied to determine its ability to generate a respiratory burst after stimulation with phorbol myristate acetate (PMA) or opsonized zymosan. U937 cells cultured in normal medium produced virtually no superoxide anion or chemiluminescence in response to either stimulus. In contrast, U937 cells cultured in medium containing soluble factors from activated lymphocytes produced significant O2- and chemiluminescence when stimulated with PMA or opsonized zymosan. The chemiluminescence in response to PMA was maximal in U937 cells precultured with these soluble factors for 3 days, whereas maximal responsiveness to opsonized zymosan was not observed until 5 to 6 days of lymphokine exposure. Although this ability to generate a respiratory burst persisted for a number of days in U937 cells that were subsequently recultured in normal medium, this responsiveness was gradually lost in the continued absence of these factors. The data indicate that the U937 monocytic cell line can be activated or induced to differentiate by soluble factors released by activated lymphocytes. In the process, these cells acquire the ability to generate a respiratory burst. The U937 cell line may serve as a useful model for the study of the ontogeny and regulation of the respiratory burst during human monocytic differentiation.

Migration of neutrophils from blood into tissue is a complex response by circulating cells to chemotactic stimulation. Previous studies of the functional changes induced by this process have produced variable results. We compared neutrophils isolated from blood and from subcutaneous wounds in rabbits using established assays for adherence, chemotaxis, superoxide anion production, and hydrogen peroxide production. No differences in adherence to biologic surfaces or chemotaxis toward activated plasma were found. However, our results confirm the observation that wound neutrophils are "primed" for increased production of oxygen radicals. Primed responses were observed for both soluble (formyl methionyl leucylphenylalanine, phorbol myristate acetate) and particulate (opsonized zymosan) stimulants. Priming was also observed for peritoneal exudate neutrophils. The data suggest that the process of extravascular migration includes priming of the superoxide generating system.

Fluoranthene, a non-carcinogenic polycyclic aromatic hydrocarbon, inactivates Escherichia coli cells in the presence of near-ultraviolet light (NUV; 300-400 nm). E coli cells carrying defects in the uvrA6 or katF genes are sensitized to inactivation by the simultaneous treatment with fluoranthene and NUV, suggesting that DNA is a target and that hydrogen peroxide is generated. Haemophilus influenzae transforming DNA can be inactivated by the simultaneous treatment with fluoranthene and NUV confirming DNA as a target. Using the photooxidation of imidazole and histidine as probes, fluoranthene was found to generate singlet oxygen in organic and aqueous media. In water, it participated in electron transfer reactions, reducing nitro blue tetrazolium as well as ferricytochrome C. This reduction took place both in the presence of air, where superoxide anion was formed, and under argon. Simultaneous treatment with fluoranthene and NUV was incapable of inducing histidine-independent mutations. Simultaneous treatment with fluoranthene and NUV was incapable of inducing the uvrA gene product as evidenced by the absence of the induction of beta-galactosidase in an E coli operon fusion strain [uvrA215::Mud(Ap,lac)].

In this study, we have shown that chickens, frogs, and toads are resistant to acute pulmonary injury by a variety of toxic agents, (O2, hyperbaric O2, paraquat, and silica), that cause extensive acute injury in mammals. Acute pulmonary injury is defined as a massive influx of inflammatory cells, both interstitially and into the alveolar spaces, pulmonary edema, hemorrhage, and the presence of H2O2 and O-2 in the lavaged supernatant, occurring within 48 h. In some cases, chronic effects of the toxins were observed after 90 h., i.e., hemorrhage, fibrosis, and an accumulation of interstitial inflammatory cells. In all three nonmammal systems, isolated inflammatory cells failed to respond chemotactically in vitro to known mammalian chemotaxins. Pulmonary lavage of the exposed chickens, frogs, and toads also failed to produce inflammatory cells. Pulmonary edema was not detected in any of the animals by comparison of lung weight to total body weight. Intratracheal injections of silica for 2 weeks did produce chronic effects in chickens and frogs. Morphologically, the lungs showed signs of fibrosis and accumulation of interstitial inflammatory cells, but no intraalveolar cells. After 90 h of hyperbaric O2, frogs exhibited a massive infiltration of interstitial inflammatory cells and hemorrhage. Elevated O2 levels (100%) for 2 weeks under normal atmospheric conditions produced no changes in frog lungs or in the amount of inflammatory cells in the lungs. Intravenous injections of paraquat for up to 208 h failed to initiate an accumulation of pulmonary inflammatory cells or the development of pulmonary edema in chickens. There was also no detectable H2O2 or O-2 in the lavaged supernatant. It was not determined whether paraquat had a longer or more chronic effect on chickens. We suggest that the lack of an acute pulmonary inflammatory mechanism in chickens, frogs, and toads is in part responsible for the resistance of these animals to acute pulmonary injury by oxidizing mammalian toxins.

In malnourished patients, little is known about production of superoxide anion which plays an important role in bactericidal activities of polymorphonuclear leukocytes (PMNs). In this study, superoxide production of RMNs was assayed in 98 malnourished patients with cancer of digestive organs, in preoperative and untreated state, in order to evaluate the bactericidal capacity, and following results were obtained. Superoxide production of PMNs in patients with cancer was significantly decreased in comparison with healthy controls, especially in advanced cancer patients. Furthermore, superoxide production of PMNs in cancer patients who were suffered from postoperative septic complications was significantly decreased in comparison with the controls and no complication group. Patients with advanced gastric cancer were evaluated as a state of malnutrition in nutritional assessment. Superoxide production of PMNs in malnourished patients with cancer of digestive organs was depressed. In gastric cancer patients, there were no differences in superoxide production of PMNs among clinical stages in well-nourished patients. On the other hand only in stage IV group of malnourished patients low values were presented. These results may suggest that the decrease in superoxide production of PMNs in patients with cancer contributes to high susceptibility to postoperative infection and is induced by malnutrition.

The generation of human PAF was examined using purified neutrophils from normal and CGD donors. PAF release occurred in response to all of the neutrophil stimuli examined, including PMA and the calcium ionophore A23187, which initiate the relatively selective release of specific granule constituents. PAF release was dissociable from neutrophil degranulation by 1) the lack of correlation between PAF titers and extent of enzyme release for different stimuli, 2) the strict dependence of PAF release on the presence of extracellular Ca++, 3) the different kinetics for release of PAF and granule enzymes, and 4) the ability of neutrophils significantly depleted of azurophil and specific granule constituents to release normal levels of PAF when stimulated with opsonized zymosan. PAF release was also normal, if not elevated, in neutrophils from patients with chronic granulomatous disease, demonstrating that the formation and release of PAF does not depend upon an intact superoxide generating pathway.

Tumor killing by human alveolar macrophages (AM) might be an important mechanism of pulmonary defense against neoplastic disease. We compared AM and blood monocytes (Mo) for the ability to kill 2 neoplastic targets, A549 human lung adenocarcinoma cells and P815 mastocytoma cells. Blood monocytes were able to kill both targets, whereas AM killed neither. Tumor killing by Mo was spontaneous and was not increased by incubation with lipopolysaccharide. Because the P815 target is highly sensitive to lysis by hydrogen peroxide (H2O2), it afforded the opportunity to compare AM and Mo for the ability to kill tumors by the production of toxic oxygen compounds. Comparable amounts of superoxide anion were produced by AM and Mo after stimulation with phorbol myristate acetate. However, luminol-enhanced chemiluminescence of AM was far less than that of Mo, suggesting that AM could not utilize the myeloperoxidase-H2O2-halide ion system for tumor killing. The addition of exogenous peroxidase to cultures of AM and P815 cells enabled AM to kill this tumor cell. Our results suggest that as Mo mature into AM, their ability to kill tumor cells declines and that AM may be unable to kill H2O2-sensitive tumors because of a loss of myeloperoxidase during maturation.

Zymosan stimulated oxygen metabolism was investigated in polymorphonuclear leucocytes (PMN) from 6 diabetic patients. Oxygen uptake and superoxide production were continuously measured in the presence of autologous or control serum and non-opsonized zymosan, or in the absence of serum and preopsonized zymosan. The only significant impairment in the diabetic cells studied was a lower oxygen uptake in the presence of autologous serum. This defect was normalized by addition of control serum or by omitting the serum and stimulating with opsonized zymosan. In the absence of serum, the oxygen consumption was markedly diminished in only one subject, whereas two subjects showed a decrease of superoxide production in the presence of control serum. An inverse correlation between fasting glucose concentration and oxygen uptake could be demonstrated. However, exposure of normal PMN to hyperglycemic glucose concentration in vitro did not significantly alter their oxygen metabolism, suggesting that glucose alone could not be the only factor responsible for the impaired oxygen consumption in diabetic cells.

The effects of levamisole, at concentrations known to stimulate neutrophil motility, on neutrophil post-phagocytic metabolic activity were investigated. Levamisole at these concentrations caused inhibition of hexose monophosphate shunt (HMS) activity, superoxide production, hydrogen peroxide generation and myeloperoxidase (MPO), mediated iodination of ingested Candida albicans,. The inhibition of MPO-mediated iodination was not solely due to lack of H2O availability as a result of decreased HMS activity but also to a primary inhibition of iodination as shown in a cell-free system with horse-radish peroxidase (HRP) and added H2O2 and sodium iodide. Further experiments were designed to investigate possible relationships between stimulation and motility and levamisole-induced inhibition of superoxide generation and peroxidase-mediated iodination. These showed that the peroxidase/halide/H2O2 system caused inactivation of both the leucoattractant and neutrophil chemotactic responsiveness. However, concentrations of levamisole, which stimulate motility and inhibit superoxide production and peroxidase-mediated iodination, protected both the leucoattractant response and the ability of the cell to respond to the leucoattractant from inactivation by the HRP/H2O2/iodide system.

Anionic amphiphiles such as long chain unsaturated fatty acids and SDS were shown to activate the superoxide (O2-) producing NADPH oxidase in a cell-free system derived from sonically disrupted phagocytes (macrophages and granulocytes). O2- production required the cooperation of a membrane associated component sedimenting at 48,000 X g (pi) and a cytosolic factor (sigma). The purpose of the present investigation was to find out whether components pi and sigma were also present in non-phagocytic cells that do not produce O2- when stimulated. It was found that the 48,000 X g pellets of guinea pig lymph node and thymus cell sonicates contained significant amounts of component pi, as shown by their ability to support SDS-elicited NADPH-dependent O2- production when supplemented with macrophage cytosol. Lymph node and thymus pi could be extracted from the membrane by 30 mM octyl glucoside, just as its macrophage-derived equivalent. Combining lymph node and thymus 48,000 X g pellet with autologous cytosol did not yield an active enzyme preparation. Also, cytosol from lymph node and thymus cells could not cooperate with macrophage 48,000 X g pellet, indicating that component sigma was lacking in lymphoid cells. Neither pi nor sigma could be detected in guinea pig kidney, the mouse myeloma cell line MOPC 315 and the canine cell line Cf2Th. The 48,000 X g pellet of all nonphagocytic cells examined contained a b-cytochrome that resembled, by its spectral characteristics, the cytochrome b559 thought to be characteristic of phagocytes. In macrophages, cytochrome b559 represented 80% of b-cytochrome content of the 48,000 X g pellet, whereas in non-phagocytic cells, the equivalent material represented only 50 to 60%. There was no correlation between the presence and quantity of the cytochrome b559-like chromophore in the 48,000 X g pellet of a particular cell type and its ability to cooperate with macrophage cytosol in SDS-elicited O2- production.

The technique of lucigenin-dependent phagocytic chemiluminescence was used to investigate the stimulation of metabolic activity in human alveolar macrophages on contact with polystyrene. The results were similar to those obtained using a spectrophotometric assay of superoxide release based on ferricytochrome C reduction. There was a marked stimulation of metabolic activity in the alveolar macrophages on incubation at 37 degrees in polystyrene vials which was shown to be due to contact with and adherence to the polystyrene. This could be reduced by the addition of gelatin or foetal calf serum without preventing the ability of the cells to respond to opsonized particles. By using several metabolic inhibitors it was shown that lucigenin-dependent chemiluminescence was associated with the release of superoxide at the time of adherence. The implications of these findings are discussed and it is suggested that the stimulation of alveolar macrophage metabolic activity by contact with polystyrene can contribute to the observed difference between alveolar macrophage and polymorphonuclear leucocyte oxygen consumption in the absence of phagocytosis.

Dnacin B1 preferentially inhibited the incorporation of [3H]thymidine into acid-insoluble fractions in Escherichia coli. At a sublethal concentration, dnacin B1 caused filamentous growth in E. coli and induced prophage lambda. The antibiotic also showed potent bactericidal activity against repair-deficient E. coli strains, such as recA, recB, and polA strains, In in vitro studies, dnacin Ba raised the melting temperatures of various double-stranded DNAs. In addition, the antibiotic showed DNA-cleaving activity against PM2 DNA in the presence of reducing agents, and the activity was suppressed by scavengers for oxygen free radicals and an iron-specific chelator, desferrioxamine E. The stimulation of the generation of superoxide radical by dnacin B1 was confirmed by measuring the reduction of neotetrazolium. Therefore, it can be presumed that the primary cellular target of dnacin B1 is DNA in susceptible cells, and the autooxidation of DNA-bound dnacin B1 causes the generation of oxygen-free radicals that result in the damage of DNA and the inhibition of its synthesis.

The pathways of action of some agents modifying tumor dissemination were studied experimentally. The preventive effect of interferon upon metastatic spreading was found to be due to activation of effector cells of non-specific immune surveillance. A relationship between tumor dissemination process and the condition of neuro-regulatory mechanisms of target-organ was established. For example, it was shown that an injury to the vascular wall of the target-organ caused by hypercatecholemia and steady rise in adrenocortical hormone levels is, to a degree, a factor of metastatic spread stimulation following primary tumor removal.

Guinea-pig peritoneal polymorphonuclear leukocytes promoted superoxide anion (O2(-)) generation when stimulated with soluble antigen-antibody complex, concanavalin A or sodium dodecyl sulfate. The enhancement with antigen-antibody complex or concanavalin A was inhibited with diisopropyl fluorophosphate. On the other hand, the enhancement with sodium dodecyl sulfate was not affected by the inhibitor. L-1-pTosylamido-2-phenylethyl chloromethyl ketone (Tos-PheCH2Cl) and tetrahydrofuran also enhanced O2(-) generation even in the presence of diisopropyl fluorophosphate, while at low concentrations they inhibited O2(-) generation with antigen-antibody complex. These results indicate that a certain diisopropyl fluorophosphate-sensitive factor may be involved in the O2(-)-generating response of leukocytes to antigen-antibody complexes or concanavalin A, but not in that to sodium dodecyl sulfate, Tos-PheCH2Cl or tetrahydrofuran.

Cultured porcine retinal pigment epithelial cells release superoxide, measured as superoxide dismutase (SOD)-suppressible reduction of cytochrome C, and SOD-suppressible luminol-dependent chemiluminescence. Latex beads stimulated a significant release of superoxide that reached 82 nmol/mg protein in the first 15 min and declined thereafter. Formation of an insoluble blue formazan following reduction of nitroblue tetrazolium histochemically demonstrated that superoxide was released over the region of the cell vicinal to the bead. Dioctanoylglycerol, a synthetic, cell-permeating activator of protein kinase C, also elicited a rapid release of superoxide from RPE cells. This study emphasizes the need to characterize the mechanisms by which superoxide is generated, whether its release is regulated, and how toxicity is prevented in vivo.

Representatives of the major classes of nonsteroidal antiinflammatory drugs (NSAID) were assessed for their effects on superoxide anion (O2-.) production by human polymorphonuclear leukocytes stimulated with phorbol myristate acetate or N-formyl methionyl-leucyl phenylalanine (fMLP). Three levels of effects were studied: (1) overall inhibition of O2-. production, (2) the inhibition of interaction between fMLP and specific receptors at the cell surface, and (3) intermediate proenzyme and enzyme effects. Some, but not all drugs inhibited O2-. production. In general, drugs that inhibited O2-. production inhibited fMLP-receptor interactions in a consistent dose dependent fashion, showing noncompetitive kinetics. Drugs that failed to inhibit O2-. production showed weak and variable effects on receptor binding and on the intermediate enzymes. Clinical observations suggest that inflammation in diseases such as gout respond differently to NSAID than diseases such as rheumatoid arthritis; studies of drug effects may help to clarify the differences in pathogenesis of these inflammatory diseases.

The mechanism by which cadmium produced oxidizing effects in vivo is unknown. We show that cadmium enhances the production of superoxide anion radical (O-(2) .), a reactive oxygen species, in digitonin-stimulated phagocytes from man and rat. Cadmium concentrations ranging from 3.6 X 10(-2)M to 3.6 X 10(-4)M inhibited O-(2) . production in rat alveolar macrophages or human granulocytes. However, when activated in the presence of 3.6 X 10(-5)M cadmium, the production of O-(2) . was increased by a factor of 2.11 +/- 0.25 above control levels in human granulocytes and 3.6 +/- 0.62 above control levels in rat alveolar macrophages. This effect by levels of cadmium within the range of those occurring during in vivo toxicity might provide an explanation for the oxidizing effects of this metal ion.

The effects of the beta-receptor blockading agents, metoprolol and sotalol on neutrophil random motility, chemotaxis, post-phagocytic glycolysis, superoxide production, hexose monophosphate shunt activity, myeloperoxidase (MPO) mediated protein iodination and hydrogen peroxide production were assessed in vitro. The concentration range investigated was 10(-8)--10(-2) M for each drug. Both agents caused significant stimulation of neutrophil motility at concentrations of more than 10(-4) M. Increased migration was not associated with increased glycolysis or significant cyclic nucleotide fluctuations, but was inversely related to inhibition of superoxide and hydrogen peroxide generation and MPO mediated iodination with both drugs. In a further series of experiments to determine the relationship between the drug induced inhibition of H2O2 production and MPO mediated protein iodination to stimulation of motility it was found that concentrations of sotalol and metoprolol that caused these effects prevented HRP/H2O2/I- induced inactivation of the leucoattractant and inhibition of neutrophil chemotactic responsiveness. Neither drug inhibited the activity of MPO per se nor the reduction of ferricytochrome c by superoxide generated by the xanthine: xanthine oxidase system in vitro. It is suggested that enhanced neutrophil motility is not related to beta-receptor blockade but rather to restricting the availability of hydrogen peroxide and reactive products of the MPO/H2O2/halide system.

Human neutrophils exposed to 10(-4) M doxorubicin and the derivatives epirubicin and thepirubicin revealed a different intracellular penetration and distribution pattern as demonstrated by fluorescence microscopy and fluorimetric determination of drug intracellular concentration. While doxorubicin was found to be a potent inducer of superoxide generation from resting cells, epirubicin exhibited less superoxide-inducing power. Thepirubicin on the contrary did not show any superoxide-inducing effect. Moreover the anthracyclines tested all inhibited the phorbol ester-stimulated chemiluminescent response to the same extent, which suggested a common target for the drug action. Anthracycline-stimulated superoxide production seems to correlate with the cardiotoxic effects. The most cardiotoxic drug, doxorubicin, is the most potent inducer of superoxide generation, while epirubicin, which is less cardiotoxic, has a relatively limited effect on superoxide production. Thepirubicin which has been shown not to induce delayed cardiomyopathy has no effect on superoxide release from the cells.

Because imidazole acetic acid (IAA), a product of histamine catabolism was shown to inhibit histaminase release from human polymorphonuclear leukocytes (PMNs), the effect of this compound on other neutrophil functions was investigated. IAA at concentrations of 10(-10) or more inhibited histaminase release induced by particle-bound C3b, the larger fragment of the activated form of the third component of complement. Release of histaminase induced by aggregated IgG, phorbal myristate acetate (PMA), formyl-methionyl-leucyl-phenylalanine (FMLP) and calcium ionophore was not affected by IAA. In addition IAA had no effect on release of beta-glucuronidase, myeloperoxidase, and lysozyme or on phagocytosis and superoxide generation. IAA did modestly inhibit neutrophil chemotaxis. These findings suggest a highly specific modulating effect of the histamine catabolite IAA on complement-mediated PMN function.

The effect of sodium aurothiomalate and auranofin on the generation of superoxide anions (O2-) by polymorphonuclear leukocytes (PMNLs) and adherent mononuclear phagocytic cells (AMNCs) has been investigated. Sodium aurothiomalate at final concentrations of 1, 10, and 100 micrograms Au/ml and auranofin ranging from 0.1 to 2.0 micrograms Au/ml were used in the reactions involving all cell types. Results have been compared between cells drawn from normal controls and patients with active rheumatoid disease. The effect of gold compounds on both cell types was assessed following activation by phorbol myristate acetate (1 X 10(-8) M) and N-formyl-methionyl-leucyl-phenylalanine (1 X 10(-4) M) using a cytochrome c reduction method. Sodium aurothiomalate at the maximum concentration modestly inhibited O2- generation by PMNLs but not AMNCs. Auranofin inhibits O2- generation by both cell types. Inhibition of cells from patients with rheumatoid arthritis was greater than that seen with cells from normal controls.

Phagocytosis, bactericidal capacity and superoxide anion production of polymorphonuclear neutrophils (PMN) were estimated in 30 patients with well-controlled insulin-dependent diabetes (IDD) and in 50 patients with non insulin-dependent diabetes (NIDD). The estimations were additionally done in 20 elderly patients without glucose intolerance. The estimations of bactericidal capacity were performed in autologous-, zymosan activated-, inactivated- and control plasma. The phagocytosis of viable staphylococci was unchanged in all evaluated groups. The bactericidal capacity in all diabetic patients was significantly reduced. It was fully correctable in patients with IDD by suspension of cells in control or zymosan activated plasma. The improvement of PMN bactericidal capacity in patients with NIDD in similar conditions was less distinct. The superoxide anion production in patients with IDD was similar to values noticed in healthy persons. Whereas, the O2- production in patients with NIDD as well as in elderly patients were significantly reduced and correlated significantly with bactericidal capacity impairment. The possible mechanism of noticed disturbances were discussed.

Recent studies have shown that in vitro exposure of peripheral blood mononuclear cells (PBMC) to morphine results in suppressed respiratory-burst activity of monocytes and impaired interferon-gamma (IFN-gamma) production by lymphocytes. To investigate the potential in vivo effect of an opiate on these cell functions, PBMC were obtained from patients maintained on methadone. These freshly isolated mononuclear cells had a significantly impaired capacity to generate superoxide anion (O2-) in response to phorbol myristate acetate (PMA), while production of IFN-gamma by concanavalin A-stimulated cells was intact. After cell culture for 48 h, the defective O2- generating capacity was sustained. Also, culturing PBMC from healthy controls in the presence of methadone or morphine at concentrations as low as 10(-12) M caused significant suppression of PMA-stimulated O2- release. Because reactive oxygen intermediates produced by PBMC may participate in host defense against opportunistic pathogens in AIDS, these results underscore the need for investigations of the biological consequences of opiate-mediated immunosuppression.

Phagocytosis and membrane perturbation in mouse macrophages results in an increased superoxide ion production which is in direct proportion to the concomitant increase in adenosine deaminase activity. Since adenosine deaminase activity controls the amount of substrate available to xanthine oxidase, and the latter produces superoxide during turnover of its substrates, it is proposed that the purine salvage pathway is an important source of the superoxide requirement of macrophages. It is further proposed that this may be the basis, at least in part, for the mechanism of the association of immunodeficiency with adenosine deaminase deficiency.

Various cationic polyelectrolytes (poly-alpha-amino acids and histones), lectins, the chemotactic peptide, f-methionyl-leucyl-phenylalanine (fMLP), the calcium ionophore A23187, and phorbol myristate acetate (PMA) were investigated regarding their capacity to induce luminol-dependent chemiluminescence (LDCL) and superoxide production by human blood leukocytes. Although when tested individually, poly-L-arginine (PARG), phytohemagglutinin (PHA), concanavalin A (Con A), or fMLP induced only a low to moderate LDCL response, very intense synergistic CL reactions were obtained by mixtures of PARG + PHA, PARG + Con A, PARG + PHA + fMLP, Ca2 + ionophore + PARG + PHA + fMLP, and PARG + PMA. The sequence of addition of the various agents to WBC in the presence of luminol absolutely determined the intensity of the LDCL signals obtained, the highest reactions being achieved when the WBC were preincubated for 2-3 min with A23187 followed by the sequential addition of fMLP, PARG, and PHA. These "multiple hits" induced CL reactions which were many times higher than those obtained by each factor alone. On the other hand, neither poly-L-lysine, poly-L-ornithine, poly-L-histidine, nor poly-L-asparagine, when employed at equimolar concentrations, cooperated efficiently with PHA and fMLP to trigger synergistic LDCL responses in leukocytes. Concomitantly with the induction of LDCL, certain ligand mixtures also triggered the production of superoxide. The LDCL which was induced by the "cocktail" of agents was markedly inhibited by sodium azide (93% inhibition), but to a lesser extent by catalase (10% inhibition) or by superoxide dismutase (20%-60% inhibition). On the other hand, scavengers of singlet oxygen and OH (sodium benzoate, histidine) did not affect the synergistic LDCL responses induced by these multiple ligands. Cytochalasin B also markedly inhibited the LDCL responses induced either by soluble stimuli or by streptococci preopsonized either with histone or with polyanethole sulfonate. The LDCL responses which were induced by mixtures of PARG and concanavalin A were also strongly inhibited by mannose, alpha-methyl mannoside, and poly-L-glutamic acid. The data suggest that the LDCL responses induced by the soluble ligands involved a myeloperoxidase-catalyzed reaction. The possible employment of "cocktails" of ligands to enhance the bactericidal effects of PMNs, macrophages, and natural killer cells on microbial cells and mammalian targets is discussed.

Studies were carried out to determine the heterogeneity of factors that affect macrophage functions using human hybridomas constructed by fusing PHA-activated human peripheral blood lymphocytes with emetine-actinomycin D-pretreated cloned human acute lymphatic leukemia cells (CEM). Three assay systems were used to investigate the activity of the macrophage migration inhibitory factor and of the macrophage activation factors for glucose consumption (MAF-G) and for O2- formation. In the culture supernatant of hybridomas and other cells, various combinations of these activities were detected. The results indicate that at least three molecules are concerned in each of these activities.

Paraquat is a broad spectrum herbicide known to be highly lethal to man and animals. Its toxicity is characterized by acute lung injury. Paraquat produces such toxic effects through the generation of the superoxide anion according to one proposed mechanism. The present experiment, methemoglobin formation was demonstrated after incubation of oxyhemoglobin with paraquat. The generation of the superoxide anion through the interaction of oxyhemoglobin with paraquat was suggested by chemiluminescence of luminol. Superoxide dismutase (SOD) and catalare inhibited methemoglobin formation. The generation of the superoxide anion is discussed in regard to methemoglobin formation by paraquat.

The effect of respiratory syncytial virus (RSV) antibody complexes on the metabolism of human neutrophils was determined by examining the generation of luminol-dependent chemiluminescence, superoxide, and thromboxane B2. Incubation of neutrophils with RSV antibody complexes resulted in a significant increase in the production of chemiluminescence. The increase in chemiluminescence appeared to be due to (1) active phagocytosis of RSV antibody complexes as evidenced by 70% inhibition with cytochalasin B (P less than 0.001) or (2) increased superoxide production as evidenced by 40% inhibition with superoxide dismutase (P less than 0.001). The generation of superoxide was confirmed by specific analysis in a superoxide dismutase-inhibitable ferricytochrome c reduction assay. Of particular importance was the observation that RSV antibody complexes induced the release of significant quantities of thromboxane B2 from neutrophils as determined by radioimmunoassay. Oxygen radicals and/or products of arachidonic acid metabolism may, in part, mediate the pathogenesis of RSV infection through direct tissue damage and bronchoconstriction.

We prepared FMoxLP by oxidation of FMLP and evaluated its ability to trigger a variety of granulocyte responses. FMoxLP was found to depolarize granulocyte membranes; increase granulocyte oxygen consumption, superoxide production, and hydrogen peroxide production; compete with FMLP for binding to granulocytes; and stimulate granulocyte chemotaxis. Against all of these granulocyte functions, FMoxLP was considerably less potent than the parent FMLP. When luminol-dependent-granulocyte chemiluminescence (CL) was studied, FMoxLP was observed to be a more potent stimulus of this response than FMLP. This increased CL activity of FMoxLP may be related to nonoxidative burst mechanisms. Our results indicate that FMoxLP retains a significant amount of the biological activity of FMLP (albeit in general less potent). The biological importance of the observed activities of FMoxLP must await further investigation.

A calcium-activated, phospholipid-dependent protein kinase (protein kinase C) was purified to near homogeneity from human polymorphonuclear leukocytes and shown to be identical to bovine protein kinase C. The Ca2+ activation of the enzyme was studied and the Ca2+ concentrations required to activate the enzyme were compared to free cytosolic Ca2+ concentrations in resting and activated polymorphonuclear leukocytes. The free calcium concentrations in the cytosol and in the enzyme assay mixture were determined using the calcium indicator quin 2. The enzyme activity was almost totally dependent upon phosphatidylserine and could be strongly activated by Ca2+ concentrations in the micromolar range, but was not activated by phosphatidylserine at Ca2+ concentrations corresponding to the intracellular free Ca2+ concentration under resting conditions. However, at similar Ca2+ concentrations (less than 2.5 X 10(-7) M) the enzyme was highly activated by phorbol 12-myristate 13-acetate (PMA) or diolein in the presence of phosphatidylserine. It was demonstrated that PMA stimulation of human polymorphonuclear leukocytes did not induce any increase in the level of the intracellular free calcium concentration. It was concluded that PMA activation of protein kinase C occurred independently of a rise in the intracellular Ca2+ concentration. K0.5 (half-maximal activation) for the PMA activation of purified protein kinase C was shown to be equivalent to the K0.5 for PMA stimulation of superoxide (O-2) production in human polymorphonuclear leukocytes, suggesting that protein kinase C is involved in activation of the NADPH oxidase. The presumed intracellular Ca2+ antagonist TMB-8 inhibited the PMA-induced superoxide production, but neither by an intracellular Ca2+ antagonism nor by a direct inhibition of protein kinase C activity.

Serum or plasma from 3 patients with C2 deficiency (C2D) and systemic lupus erythematosus (SLE) significantly enhanced chemiluminescence and superoxide anion production by polymorphonuclear leukocytes (PMN) after stimulation with phorbol myristate acetate or latex beads. PMN from patients and normal individuals were supranormally activated when resuspended in plasma from these patients. No such effect was seen with plasma from a patient with C2D but with no evidence of SLE, from patients with SLE but not C2D, from patients with C1q or C8 deficiency, from C4-deficient guinea pigs, or NZB-NZW mice. Because oxygen-derived free radicals may cause joint or tissue damage, C2D patients who have or develop this activity in their plasma may be more prone to SLE or other collagen-vascular diseases.

The human acute promyelocytic leukemia cell line HL-60 is induced by retinoic acid (RA) and N,N-dimethylformamide (DMF) to differentiate into cells having many of the functional and morphologic characteristics of mature granulocytes. With normal human phagocytic cells there is both superoxide anion (O2-) production and chemotaxis in response to chemoattractants such as N-formyl-methionyl-leucyl-phenylalanine (FMLP). We have now found that although HL-60 cells induced with RA alone produce O2- in response to 12-0-tetradecanoyl-phorbol-13-acetate (TPA) they are deficient in FMLP-stimulated O2- production and chemotaxis. In contrast, HL-60 induced either with DMF or with a combination of 10 nmol/L RA and a T cell-derived lymphokine, differentiation-inducing activity (DIA), produce O2- and exhibit chemotaxis in response to FMLP. The basis for these results appears to be the concentration of cell surface chemotactic peptide receptors. Thus, untreated HL-60 and HL-60 induced with either RA alone or DIA alone do not have measurable levels of FMLP receptors, whereas HL-60 induced with a combination of RA and DIA has 5,400 receptors per cell. HL-60 induced with RA and DIA plus 1 mumol/L dexamethasone have 25,000 receptors per cell and have greater chemotactic activity than HL-60 induced with the combination of RA and DIA. Thus, differentiation of HL-60 to cells with many properties of normal phagocytes can be induced in vitro by physiologic substances.