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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.

The effects of clofazimine on phagocyte functions associated with antimicrobial activity have been investigated. Clofazimine at a variety of concentrations was capable of enhancing the spontaneous production of hydrogen peroxide and the intracellular killing ability of phagocytes; but had no effect on resting phagocyte lysozyme release, or hexose monophosphate shunt (HMPS) activity. However, when these latter functions were assessed in the presence of a phagocytic stimulus, clofazimine moderately increased both lysozyme release and HMPS activity. A 25 Kd glyco-lipoprotein derived from Mycobacterium tuberculosis has been shown to inhibit these antimicrobial functions. Clofazimine was capable of partially reversing the inhibitory effect of the mycobacterial component in all of the systems assessed. Partial restoration was observed at concentrations of 0.5 mg/l and was maximal at 2 mg/l. These studies indicate important mechanisms operative in the pathogenesis of tuberculosis and suggest that clofazimine may have clinical relevance in the treatment of mycobacterial diseases.

The functional abilities of macrophages from cerebrospinal fluid (CSF) have so far been little studied. We examined the acquisition of activation characteristics by CSF macrophages during the course of experimental cryptococcal meningitis. CSF macrophages developed the ability for increased reactive oxidative intermediate (H2O2) production and tumor and fungal cytotoxicity. Despite having been activated, CSF macrophages could not inhibit the growth of Cryptococcus neoformans in vitro. Immunosuppression with cyclosporine, which eliminates the natural resistance of rabbits to cryptococcal meningitis, did not prevent or diminish H2O2 production by CSF macrophages but did reduce their tumoricidal activity. Activation of CSF macrophages appears to be an integral part of the central nervous system immune response to C. neoformans in this model, but alone is insufficient to eliminate C. neoformans from the central nervous system.

We have demonstrated previously that Chlamydia psittaci grows well in human monocyte-derived macrophages, but to a limited extent in lymphokine-or interferon-gamma (IFN-gamma)-activated macrophages. In this investigation, freshly explanted human monocytes inhibited chlamydial inclusion formation by 85% as compared to macrophages, and the level of inhibition was similar to that exhibited by lymphokine-activated macrophages (79%). To determine whether the oxygen-dependent antimicrobial mechanisms of the mononuclear phagocyte were involved in the inhibition, cells were infected with C. psittaci in the presence of agents that either inhibit the respiratory burst (glucose deprivation) or diminish the effect of H2O2 (catalase). These treatments had no effect on the capacity of monocytes and lymphokine-activated macrophages to restrict chlamydial growth. In addition, monocytes and activated macrophages from an individual with chronic granulomatous disease suppressed chlamydial growth as effectively as normal cells. Oxidatively deficient HeLa and endothelial cells, once stimulated by lymphokine, also displayed normal levels of antichlamydial activity. The induction of this apparently oxygen-independent antichlamydial effect by lymphokine was completely neutralized by a monoclonal anti-IFN-gamma antibody, and could be achieved by treatment with recombinant (r)IFN-gamma alone. These results indicate that the primary antimicrobial mechanism of the human monocyte against C. psittaci is oxygen-independent, and that this response can be effectively stimulated in the macrophage by lymphokine (IFN-gamma).

We report the identification and purification of a new inflammatory monokine synthesized by the macrophage tumor cell line RAW 264.7 in response to endotoxin. This monokine, which we term "macrophage inflammatory protein" (MIP), is a doublet with an apparent molecular mass of approximately 8,000 daltons on SDS-PAGE but forms aggregates of greater than 2 x 10(6) daltons as assessed by gel filtration. Partial NH2-terminal amino acid sequence data reveal no significant homology with any previously described protein. Although the monokine is anionic under physiological conditions, it is one of two major macrophage-secreted proteins that bind to heparin at high salt concentrations. At 100 ng/ml or greater, MIP is chemokinetic for human polymorphonuclear cells and triggers hydrogen peroxide production. Subcutaneous injection of 10 ng or greater of MIP into footpads of C3H/HeJ mice elicits an inflammatory response, characterized by neutrophil infiltration. These findings suggest that MIP is an endogenous mediator that may play a role in the host responses that occur during endotoxemia and other inflammatory events.

We have previously demonstrated that a high percentage of rat cultured mesangial cells phagocytized serum-treated zymosan (STZ) (L. Baud, J. Hagege, J. Sraer, E. Rondeau, J. Perez, and R. Ardaillou, J. Exp. Med. 158: 1836-1852, 1983). Phagocytosis resulted in stimulation of arachidonic acid metabolism with generation of H2O2. Exposure of mesangial cells to dexamethasone for 48 h produced a dose-dependent decrease in phagocytosis-induced production of H2O2 with a 50% inhibitory concentration of 32 nM. The decrease in H2O2 release was associated with the inhibition of prostaglandin (PG) E2 production. The effect of dexamethasone could be considered as due to receptor-mediated modulation of protein synthesis since dexamethasone was not active immediately but only after a lag period of 3 h; RU 38486, a potent competitor for dexamethasone receptors, counteracted the reduction in H2O2 generation; and actinomycin and cycloheximide both blunted the inhibitory effect of dexamethasone. Pretreatment of mesangial cells with dexamethasone also produced a dose-dependent decrease in the phagocytic capability of the cells (63% inhibition for 1 microM dexamethasone). However, the inhibitory effect of dexamethasone on H2O2 production expressed as percentage of control was similar whether or not phagocytosis had been blocked by cytochalasin B. This result and also the fact that dexamethasone inhibited H2O2 production in cells triggered with soluble stimuli (A 23187 ionophore, PAF) suggested that the effect of dexamethasone on H2O2 generation was independent of that on phagocytosis. Addition of exogenous arachidonic acid reduced the effect of dexamethasone only when its conversion into PGE2 was inhibited by indomethacin.(ABSTRACT TRUNCATED AT 250 WORDS)

Human red blood cells were loaded with Glucose oxidase from Aspergillus niger by a standardized procedure of encapsulation involving transient hypotonic hemolysis followed by isotonic resealing. The amount of loaded enzyme activity, as evaluated by O2 consumption at 5 mM glucose, ranged from 40 to 75 mumoles O2/hr/ml of packed red cells at 37 degrees C. The red cells loaded with Glucose oxidase were found to behave as efficient glucose-consuming bioreactors. Moreover, at 5 mM glucose, no clear mechanism of H2O2-induced damage was apparent, with the exception of a significantly increased formation of methemoglobin (10% approximately) and of a several-fold stimulated intracellular rate of hexose monophosphate shunt activity, indicating glutathione peroxidase-mediated draining of reduced glutathione for removal of bursts of H2O2. The Glucose oxidase-loaded red cells represent a convenient model system for cytotoxicity studies aiming at clarifying the effects of intracellularly formed H2O2.

A study was made of a lymphokine produced by human T lymphocytes that mediates activation of human monocytes and monocyte-like cell lines, measured by increased production of H2O2. The lymphokine was produced either by stimulation of human nonadherent peripheral blood mononuclear cells with concanavalin A (Con A) or by stimulation of a human T cell line, HSB2, with Con A and phorbol myristic acetate (PMA). When incubated with freshly isolated peripheral blood monocytes for 48 to 72 hr, the H2O2-potentiating factor (HPPF) stimulated increased production of H2O2, measured in a PMA-triggered assay for H2O2 secretion. Because variations occurred in the response of normal blood donors to the HPPF, human monocyte-like cell lines were used as homogeneous and consistently responsive targets for the lymphokine to facilitate biochemical characterization studies of the factor. Two cell lines were studied: HL60, a human promyelocytic cell line, and U937, a human histiocytic cell line. When target cells of either type were incubated in the presence of the HPPF for 48 to 72 hr, they produced increased amounts of H2O2 in a dose-dependent fashion. H2O2 levels were assessed by means of a microassay that measures peroxide-mediated oxidation of phenol red after an oxidative burst triggered with PMA. By using this assay, HPPF was found to have an apparent m.w. of 54,000 and an isoelectric point of 5.5. The bouyant density was determined to be 1.307, indicating that HPPF is a protein. The utilization of cell lines for both the production and assay of HPPF should facilitate the purification of this lymphokine and the subsequent evaluation of its relationship to other lymphokines known to affect macrophage microbicidal and tumoricidal function.

Microorganisms ingested by PMNs are exposed to a variety of antimicrobial systems. Together they comprise a formidable armamentarium, and few organisms survive. The predominant antimicrobial system would be expected to vary with the species, the availability of oxygen and the type of microorganism ingested. There is considerable evidence that the MPO-mediated antimicrobial system plays an important role in the destruction of certain microorganisms in most species; chicken heterophils, however, do not contain MPO,40 and some microorganisms are resistant to this system due to the nature of their cell wall material.146 Further, microbial catalase may offer some protection. The granulocytes of some species (e.g., rabbit, chicken) are rich in cationic proteins and these agents may play a particularly important role in these cells. Granular cationic proteins are less plentiful in human cells.111 Organisms vary in their susceptibility to lysozyme and this enzyme is absent from bovine leukocytes.113 It is probable that the total microbicidal potential of the leukocyte is in excess of its needs under most circumstances. This "overkill" capacity is a reflection of both the level of activity of individual systems and their variety. Particular organisms are susceptible to more than one antimicrobial system and thus may be effectively handled by back-up systems when one is absent. Thus, an organism normally killed by the peroxidase system may be handled less efficiently but adequately when MPO is absent by other oxygen-dependent antimicrobial systems. When a defect in oxidative metabolixm is present as in CGD, both MPO-catalyzed and nonenzymatic oxygen-dependent systems are absent. The ingested organism can, in some instances, supply the needed product of oxidative metabolism (i.e., H2O2); in other instances, oxygen-independent antimicrobial systems are adequate to prevent microbial growth. However, in yet other instances, the organisms survive and multiply and severe infection results.

The effect of 6-O-QS-10-N-acetylmuramyl-L-valyl-D-isoglutamine methyl ester (quinonyl-MDP-66) on various functions of macrophages was examined. Mouse peritoneal macrophages, when treated either in vitro or in vivo with quinonyl-MDP-66 suspended in phosphate-buffered saline, showed a capacity for cytolysis and cytostasis against tumor targets and released H2O2 in the presence of phorbol myristate acetate. The macrophages induced by quinonyl-MDP-66 also had both antibody-dependent cell-mediated cytotoxicity and phagocytic activity against erythroid targets. The fact that synthetic quinonyl-MDP-66 stimulates the macrophages to become more cytotoxic than do other MDP analogs suggests that the lipophilic residue (QS-10) in quinonyl-MDP-66 may be important for the development of this activity.

Generation of H2O2 by rat brain mitochondria using succinate and glycerol-1-phosphate as substrates has been demonstrated. Earlier workers were unable to detect this activity in sucrose-Tris buffer. We found that this was due to a lag in the expression of activity in sucrose medium. Using phosphate buffer (50 mM), good rates are now obtained. Generation of H2O2 by rat brain mitochondria required the presence of antimycin A and was dependent on the substrates succinate and glycerol-1-phosphate. Low rates were obtained with NAD+-linked substrates and none with choline, glutamate, and NADH. The Km and Vmax values for H2O2 generation were considerably lower than the corresponding values for the respective dehydrogenase activity, measured by dye reduction. Oxygen-radical scavengers inhibited H2O2 generation, suggesting oxygen radical involvement. Depletion of ubiquinone from mitochondria resulted in loss of H2O2 generation. Reconstitution of such depleted particles with ubiquinone restored the capacity to generate H2O2 in a concentration-dependent manner. Levels of H2O2 production were found to be maximal in cerebellum. Brain mitochondria from rabbit, hamster, mouse, and guinea pig also have the capacity to generate H2O2 on oxidation of glycerol-1-phosphate.

beta-Lapachone, an antimicrobial agent, markedly increase the generation of H2O2 in intact Trypanosoma cruzi epimastigotes (Y strain). Increase in H2O2 was determined by the horseradish peroxidase-H2O2 Compound II formation as well as by a cytochemical technique.

We used isolated, buffer-perfused rabbit hearts to evaluate whether global, normothermic ischemia altered mitochondrial hydrogen peroxide (H2O2) generation and mitochondrial activities of the major enzymes responsible for degrading H2O2 and superoxide anion (O2-.): glutathione peroxidase (GPD) and superoxide dismutase (SOD), respectively. This preparation lacks exogenous neutrophils and endogenous xanthine oxidase, which are other potential sources of oxygen metabolites. Ischemia depressed mitochondrial oxidative phosphorylation parameters, State 4 succinate-supported H2O2 generation rates, and the relative flux of State 4 oxygen consumption that was diverted to H2O2 formation. The production of H2O2 was not abolished. Ischemia and reperfusion significantly reduced the activities of SOD (by 43%) and GPD (by 39%) in the mitochondrial fraction. Cytosolic GPD activity was also depressed. The results suggest that the myocardial cell's ability to enzymatically degrade H2O2 and O2-. is compromised, particularly in the mitochondrion. Although mitochondrial H2O2 production is decreased, the mitochondria may persist as a source of this oxygen metabolite following ischemia. Collectively, the data may help explain why mitochondria are vulnerable targets of free radical-mediated damage due to ischemia.

A large number of human mononuclear cells were simultaneously separated into fractions enriched in B cells, T cells, large granular lymphocytes (LGL) and monocytes by centrifugal elutriation. Lymphocyte populations were analyzed using monoclonal antibodies. In particular, highly enriched natural killer cells, Leu7+ cells, were collected in the intermediate fractions. Monocytes, which were identified as esterase positive cells, and Leu M3 cells were collected at higher counterflow rates and in the final fraction. The purity of monocytes in the final fraction was 81%. The oxidative metabolic activity (H2O2 production) and non-specific esterase activity of individual monocytes was estimated in the analysis of functional heterogeneity of monocytes using flow cytometry. 2',7'-dichlorofluorescein diacetate (DCFH-DA) and fluorescein diacetate (FDA) were used as indicators in the measurement of H2O2 generation and esterase activity. Intracellular generation of a fluorescence product (H2O2 Production; average percentage of fluorescence positive cells) of monocytes in the stimulation of phorbol myristate acetate (PMA, 100 ng/ml) was greater in larger than smaller cells. H2O2 production gradually increased from 6% and 25-38% and 60% in the intermediate and final fractions respectively. Furthermore, the average fluorescence intensity of the large monocyte population in the final fraction was 1.13-1.31 fold more active than that of the smaller cells. Thus, the functional heterogeneity of human monocytes was further confirmed in the assays of H2O2 production exposed to PMA and FDA hydrolysis using flow cytometry. Furthermore, the CCE system can isolate lymphocyte subsets and LGL.

It seems that superoxide dismutase plays the key role in protecting aerobes against O2 toxicity, but there is a whole range of ancillary mechanisms: enzymes to remove H2O2 (catalase, peroxidases) and hence to control formation of .OH from O2, which requires H2O2; antioxidants (ascorbate, GSH, alpha-tocopherol, carotenoids), which also react with singlet oxygen and/or .OH and often inhibit lipid peroxidation and last, but not least in animals, glutathione peroxidase, which controls the rate of lipid peroxidation. These mechanisms cope well at normal O2 concentrations but are insufficient at higher levels.

The rates of the NADPH-dependent formation of superoxide radicals and hydrogen peroxide have been measured in liver microsomes from phenobarbital-pretreated rats. Correcting a quenching of O2(-) radicals by microsomes, a stoichiometry of O2(-) to H2O2 close to 2:1 was obtained. This, and the fact that pseudo-substrates of microsomal cytochrome P450 like perfluoro-n-hexane and perfluorinated cyclohexane did not increase H2O2 formation in a catalase-inhibited assay, rules out a two-electron reduced oxygen species as the source of H2O2. The rates of O2(-) as well as H2O2 generation in the presence of 7-ethoxycoumarin were equally inhibited by carbon monoxide (75%) and resulted in photochemical action spectra with a maximum reactivation at 450 nm. Using the same conditions the monooxygenation was inhibited to a high degree (83%) but without exogenous substrate the inhibition of H2O2 formation dropped to 55%. It was concluded that most of the O2(-) originated from the oxycomplex of cytochrome P450 and that substrates can modify the rates of its decomposition and sensitivity to carbon monoxide. No correlation of H2O2 formation or of substrate monooxygenation with the optical substrate binding spectra could be observed. From the pH dependence a proton-assisted decomposition of oxy-cytochrome P450 appears likely. H2O2 formation was only slightly decreased at 20 microM dioxygen suggesting that H2O2 formation via cytochrome P450 should also occur in vivo.

To investigate the specific nature and timing of oxygen (O2) metabolite reperfusion injury, we used a rat-heart model (Langendorff's solution, 37 degrees C) and hydrogen peroxide (H2O2)-dependent aminotriazole inactivation of catalase as a measure of myocardial H2O2 before, during, and after ischemia. We found that after ischemia (20 minutes, global, 37 degrees C), ventricular functional loss--as assessed by measurement of developed pressure (DP), +dp/dt, and -dp/dt with a ventricular balloon--occurred at 10 minutes of reperfusion and that myocardial H2O2 production was maximal by this time. Furthermore, H2O2 production did not occur during ischemia, and inhibition of xanthine oxidase by tungsten feeding or infusing a permeable O2 metabolite scavenger during reperfusion (dimethylthiourea) prevented ventricular functional loss. We conclude that (1) reperfusion injury is in part mediated by toxic oxygen metabolites, (2) H2O2 is the central O2 metabolite responsible for reperfusion injury, and (3) the timing of H2O2 production coincides with the timing of ventricular functional loss.

Listeria monocytogenes, a gram-positive motile bacterium which can cause severe bacterial infection in humans, is considered to be pathogenic by virtue of its ability to resist intracellular killing. Since the mechanism of intracellular survival is poorly understood, we assessed the sensitivity of L. monocytogenes to several potent antibacterial products. Phorbol myristate acetate (PMA)-stimulated polymorphonuclear cells (PMNs) produced extracellular antibacterial products which were inhibited completely by catalase, suggesting a role for oxidative agents in this process. L. monocytogenes in logarithmic (log) growth phase resisted PMA-stimulated PMN extracellular products significantly more than L. monocytogenes in stationary (stat) growth phase or Escherichia coli (three strains) in either phase of growth. The role of oxidative agents was studied further by using xanthine oxidase-xanthine, glucose oxidase-glucose, and myeloperoxidase enzyme systems to generate hydroxyl radical (.OH), hydrogen peroxide (H2O2), and hypochlorous acid (OCl-), respectively. L. monocytogenes in log phase resisted the antibacterial products of these enzyme systems under conditions which produced superoxide (O2-) and H2O2 at concentrations similar to those produced extracellularly by PMA-stimulated PMNs, while stat-growth-phase L. monocytogenes and E. coli in either phase of growth were susceptible. Antibacterial activity could be blocked or inhibited by exogenous catalase (for all oxygen radical-generating systems), mannitol, or desferoxamine (for xanthine oxidase-xanthine) and alanine (for myeloperoxidase), suggesting that .OH and OCl- were responsible for this activity. Log-phase L. monocytogenes had 2.5-fold higher bacteria-associated catalase activity, as compared with stat-phase L. monocytogenes. These experiments, therefore, suggest that log-phase L. monocytogenes resists oxidative antibacterial agents by producing sufficient catalase to inactivate these products. This may contribute to the ability of L. monocytogenes to survive intracellularly.

Interleukin 2 (IL2) is a lymphocyte product recognised for its role as a T lymphocyte growth factor. Since some other lymphokines and monokines can modulate the function of granulocytes we examined the effects of natural and recombinant human IL2 on neutrophil locomotion, respiratory burst and degranulation. Purified T cell-derived IL2 inhibited both random and chemotactically-directed migration of neutrophils. IL2 induced a respiratory burst and release of lysosomal enzymes in neutrophils and increased these responses in classically stimulated neutrophils. Recombinant IL2 was also effective in altering neutrophil functions. We conclude that IL2 modulates neutrophil function.

Conjugates of antibody with glucose oxidase obtained via the carbohydrate moiety of the enzyme via oxidation with periodate are suggested as a tool for selective killing of the target cell. The conjugate was separated from uncoupled enzyme by repeated precipitation with ammonium sulfate (42% saturation). Purity of the conjugate was estimated by gel filtration on Toya Pearl TP-65. The glucose oxidase conjugated with rabbit antibody against mouse IgG bound specifically to plastic-adsorbed mouse immunoglobulins and to cultured human endothelial cells pretreated with mouse anti-endothelial antiserum. Glucose oxidase targeted to the cells generates hydrogen peroxide in the presence of glucose. This hydrogen peroxide killed the endothelial cells even in the absence of a halide-peroxidase system and in the presence of catalase. Features of the conjugate (specificity, effective cytotoxicity, high stability) make it suitable for prospective in vivo application and for immunoselective segregation of heterogeneous cell populations.

Exposure of human fibroblasts (IMR-90) to cool-white fluorescent light causes chromatid breaks and exchanges. This chromatid damage is caused largely by the production of hydrogen peroxide (H2O2) since it can be prevented almost completely by the addition of catalase. In support of this conclusion, exogenous H2O2 is shown to induce chromatid breaks. The clastogenic amounts of H2O2 generated during light exposure are formed within the cell since cells illuminated in saline showed the same extent of damage as cells in culture medium. Addition of selenite to the cultures during light exposure significantly decreases the chromatid damage in a dose-related manner and may be necessary to maintain sufficient activity of glutathione peroxidase. The free hydroxyl radical, . OH, appears to be partially responsible for the light-induced chromatid damage. Of the free-radical scavengers tested, i.e., mannitol, vitamin E, and dimethyl sulfoxide, only mannitol, which scavenges . OH, significantly decreases the light-induced chromatid damage. Thus, both . OH and H2O2 formed within the cell during light exposure are agents that directly or indirectly cause chromatid damage.

Previous study has shown that niridazole (NDZ) is dysmorphogenic to rat embryos between days 10 and 11 under culture conditions including 5% oxygen. Other studies have found that reductive embryonic biotransformation is required but that covalent binding is not a major basis of this embryotoxicity. In research presented here, NDZ exposure of homogenates prepared from day 10 rat embryos resulted in stimulation of oxygen uptake from incubation media. Further studies showed that a large percentage of this increased oxygen uptake was associated with the generation of superoxide anion radical and hydrogen peroxide. These findings led us to hypothesize that redox cycling forms the basis of the in vitro dysmorphogenicity of NDZ. The basic premise of this hypothesis is that as a result of redox cycling, oxygen is depleted from the sensitive tissues of embryos. In order to investigate it, we devised a technique for carefully controlling and monitoring oxygen tensions in embryo cultures. We found that when oxygen concentrations of 4% were established, a highly significant incidence of asymmetric defects resulted. These defects appeared analogous to those induced by NDZ exposure, consisting of asymmetric necrosis of mesenchymal tissue near the cephalic end of the neural tube and thinning of the neuroepithelium on the right. We concluded that the hypoxia induced by redox cycling of NDZ and related nitroheterocycles represents a major embryotoxic principle of action.

When human PMN were plated on fetal calf serum-coated polystyrene surfaces, addition of TNF-alpha, FMLP or PMA elicited adhesion and H2O2 formation. These effects of TNF-alpha and FMLP, but not of PMA, were impaired by removal of extracellular Ca2+. In addition, H2O2 formation induced by FMLP but not by TNF-alpha or PMA was inhibited by prior treatment with pertussis toxin (250-500 ng/ml). Thus, although the sequelae of TNF-alpha-receptor interaction on human PMN remain to be characterized in detail, they do not involve a pertussis toxin-sensitive GTP-binding protein.

The pulmonary alveolar macrophage (PAM) occupies a unique environment, with exposure to both the atmosphere and the blood stream. This position enables the PAM to perform its primary role of protecting the lung from inhaled, potentially damaging, agents. The effects of long term cigarette smoking on the human PAM appear to be functionally minimal when the cells are studied as a group, but the marked recruitment of PAM's which occurs in smokers leads to a greater potential for PAM helpful and/or damaging activity within the lung. The traditional role of phagocytosis is not a global function of the unstimulated human PAM. Of increasing importance for the understanding of pulmonary disease is the ability of the PAM to modulate lymphocyte, polymorphonuclear leukocyte and fibroblast activity. These functions are primarily for the defence of the lung but often produce temporary or permanent lung damage. Subclassification of the human PAM based upon functional heterogeneity may lead to a better understanding of the role of this cell.

When polymorphonuclear leukocytes (PMNL) interact with the soluble stimuli FMLP or PMA, the cells increase their production of oxidative metabolites. This increased production can be measured as luminol amplified light emission or chemiluminescence (CL). The chemiluminescence of human PMNL has been investigated, and it was found that the chemoattractant FMLP induced a bimodal response with a sharp peak of activity within 1 min, and a second peak after around 5 min. In contrast, PMA induced a one peak response reaching a maximum around 15 min after stimulis addition. Despite the fact that strictly standarized conditions for cell preparation and CL measurements were used, an extensive variability, especially in the response to FMLP was observed. Dismutation of O(2) by the addition of superoxide dismutase (SOD) or consumption of H2O2 by addition of catalase resulted in very small reductions of the CL compared to the effects on cytochrome c reduction and scopoletine fluorescence, respectively. Furthermore, since SOD reduced also the CL generated from a cell-free peroxide-peroxidase system, the specificity of SOD in the CL reaction could be questioned. Expression of CL and the effects of SOD and catalase was furthermore found to be dependent on the number of responding cells. Analysis of the effects of SOD and catalase on the bimodal FMLP response show that the first peak is strongly inhibited, whereas a very small effect upon the expression of the second peak is obtained. These results indicate, that since SOD and catalase are expected to reduce only extracellularly generated oxidative metabolites, the first peak of the FMLP response is of extracellular origin, whereas the second peak and most of the PMA induced response are cell associated or intracellular phenomena.

Among several hydroxylated metabolites of emodin, a fungal anthraquinone and constituent of rhubarb, 2-hydroxyemodin was a direct-acting mutagen showing a large electron-spin resonance (ESR) signal in the presence of DNA, especially at alkaline pH. Coupled with generation of free radical, hydrogen peroxide but not superoxide was formed. The active oxygen produced from 2-hydroxyemodin induced strand breaks in phi X 174 replicative form I DNA (supercoiled covalently closed circular duplex DNA). These results suggest a possible role of active oxygen in the process of mutagenesis.

Human recombinant tumor necrosis factor (TNF) was shown to be a weak direct stimulus of the neutrophil respiratory burst and degranulation. The stimulation, as measured by iodination, H2O2 production, and lysozyme release, was considerably increased by the presence of unopsonized zymosan in the reaction mixture, an effect which was associated with the increased ingestion of the zymosan. TNF does not act as an opsonin but, rather, reacts with the neutrophil to increase its phagocytic activity. TNF-dependent phagocytosis, as measured indirectly by iodination, is inhibited by monoclonal antibodies (Mab) 60.1 and 60.3, which recognize different epitopes on the C3bi receptor/adherence-promoting surface glycoprotein of neutrophils. Other neutrophil stimulants, namely N-formyl-methionyl-leucyl-phenylalanine, the Ca2+ ionophore A23187, and phorbol myristic acetate, also increase iodination in the presence of zymosan; as with TNF, the effect of these stimulants is inhibited by Mab 60.1 and 60.3, whereas, in contrast to that of TNF, their stimulation of iodination is unaffected by an Mab directed against TNF. TNF may be a natural stimulant of neutrophils which promotes adherence to endothelial cells and to particles, leading to increased phagocytosis, respiratory burst activity, and degranulation.

Human myeloid leukemia cells respond to various signals by differentiating to more mature cells. This study was designed to evaluate the effects of a mononuclear phagocyte-derived factor, tumor necrosis factor/cachectin (TNF), on the proliferation and differentiation of the human cell lines HL-60 (promyelocytic) and U937 (monoblastic), and to characterize TNF receptors on these cells. TNF had no effect on HL-60 cell growth or thymidine incorporation, but it markedly inhibited that of U937 cells. HL-60 cells treated with TNF formed osteoclast-like polykaryons and developed nonspecific esterase positivity. In a dose-dependent fashion, TNF enhanced HL-60 cell nonspecific esterase activity, H2O2 production, NBT reduction, and acid phosphatase content. Together, TNF and interferon-gamma (IFN-gamma) additively and synergistically caused increases in these activities as well as the expression of HLA-DR and the monocyte antigens LeuM3 (CDw14) and OKM1 (CD11). TNF also synergistically enhanced the differentiating effects of 1,25-dihydroxyvitamin D3. The potentiating actions of D3 of IFN-gamma on the TNF effect were maximal when the two agents were present together throughout the incubation, and pretreatment with TNF augmented the subsequent response to D3, but not IFN-gamma. HL-60 and U937 cells bound 125I-labeled TNF specifically, rapidly, and reversibly with binding constants of 227 and 333 pmol/L and receptors per cell of 4,435 and 6,806 for HL-60 and U937, respectively. Scatchard plots were linear, which suggested single classes of receptors. HL-60 TNF receptors were not changed by a three-day treatment with IFN-gamma or D3. U937 and HL-60 cells internalized and degraded 125I-labeled TNF to comparable degrees. TNF has differing effects on HL-60 and U937 cells that are apparently mediated through comparable high-affinity TNF receptors. The unique responses of different cell types to TNF may be due to postreceptor factors.

A simple and sensitive method for measurement of the release of H2O2 from phagocytic cells is described. The assay is based on the H2O2-dependent oxidation of homovanillic acid (3-methoxy-4-hydroxyphenylacetic acid, HVA) to a highly fluorescent dimer (2,2'-dihydroxy-3,3'-dimethoxydiphenyl-5,5'-diacetic acid) which is mediated by horse-radish peroxidase. A linear relationship between fluorescence (lambda ex = 312 nm and lambda em = 420 nm) and amount of H2O2 was found in the range of 0.1-10 nmoles per 2.25 ml assay. The method was reliable for monitoring H2O2 production in large numbers of cell samples, as suspensions or monolayers, over periods of time extending between minutes and several hours. At concentrations optimal for detection of cellular release of H2O2, HVA and horse-radish peroxidase were devoid of cytotoxic effects. The time course of H2O2 release by mouse peritoneal and bone marrow-derived macrophages and by human neutrophils was determined following stimulation with zymosan particles or phorbol myristate acetate, and the dependence of H2O2 release on cell number and stimulus dosage was studied.

Previous work from our laboratory has shown that 14-iodo-15-hydroxy-5,8,11-eicosatrienoic acid (I-HO-A) is a potent inhibitor of iodine organification in calf thyroid slices. The present studies were performed in order to clarify the mechanism of this action. Incubation of thyroid slices with 10(-4)M I-HO-A caused a 47 and 53% decrease in PB125I formation after 30 and 60 min incubation, respectively. In a series of experiments an inverse relationship between the degree of inhibition caused by I-HO-A and total iodine content and basal iodoprotein formation was observed. Chromatographic analysis of the labeled compounds showed a significant decrease in 125I incorporation into MIT, DIT, T3 and total iodolipid. The site of the inhibitory effect of I-HO-A was then sought. TPO was measured by three different methods. When TPO was solubilized from I-HO-A treated slices, no change in enzymatic activity was observed. Moreover, the same lack of action was found when solubilized TPO was incubated with I-HO-A. The production and release of H2O2 into the incubation medium was measured by chemiluminiscence technique. In control slices the values increased during the first 10 min and reached a plateau. Pretreatment of the slices with 10(-4)M KI caused a 51% inhibition, while the same concentration of I-HO-A produced a 59% inhibition. The possibility that I-HO-A might exert its action through a putative protein inhibitor was also explored. Incubation of slices with 10(-5)M I-HO-A caused a 46% decrease in PB125I formation and addition of actinomycin D or puromycin failed to alter this effect.(ABSTRACT TRUNCATED AT 250 WORDS)

The conjugation of glucose oxidase with anti-collagen antibody using periodate oxidation of the enzyme carbohydrate moiety is described. After conjugation, the antibody retained its antigen-binding capacity and the enzyme retained hydrogen peroxide-generating activity. Intradermal administration of the immune conjugate into rats induced local tissue injury at doses 10-100 micrograms. Pronounced damage (local hyperemia and edema) occurred 24 h after injection and necrosis developed 1 week later. The enzyme was tightly bound to the fibrillar components of the extracellular matrix and retained its activity in vivo for a prolonged period of time. In contrast, non-immune IgG-conjugated glucose oxidase was removed rapidly from the site of injection and did not induce tissue damage. Pure native anti-collagen antibody was retained at the site of injection for 8 days, but caused no tissue injury. These results suggest that active glucose oxidase conjugated with antibodies to tissue antigen can be accumulated and retained in the tissues. At the site of accumulation local 'proinflammatory' damage develops even in the absence of the halide-peroxidase system. Similar conjugates could be potential agents for local modulation of inflammation.

The spontaneous emission of chemiluminescence by Trypanosoma cruzi epimastigotes was 133 +/- 5 counts s-1 (mg protein)-1. The measured intracellular steady state concentration of hydrogen peroxide in the same cells was 1.5 +/- 0.5 microM. These two values are about 12- and 15-times higher than the corresponding ones for isolated rat hepatocytes. The intracellular steady state concentrations of superoxide radical and hydrogen peroxide were apparently increased by inhibiting superoxide dismutase (with diethyldithiocarbamate or KCN addition) and by the addition of two different trypanocidal agents (beta-lapachone and nifurtimox) capable of intracellular redox cycling and in each case an increased chemiluminescence was observed. Depletion of intracellular reduced non-protein SH groups by 80% increased 3-fold the chemiluminescence of T. cruzi cells. It is apparent that both an increase in the intracellular steady state concentration of superoxide anion or hydrogen peroxide and a decrease in the level of reduced SH groups lead to an increase in the level of peroxy radicals which are the precursor species for light emission.

In efforts to elucidate the role of phagocytes in host defense upon bacterial infection, we investigated the conditions necessary for induction of efficient phagocytic response together with the oxygen metabolism of the cells. The following evidence was found: (1) To induce efficient response of the phagocytes, physical impact of bacteria with the cells is required. (2) Neutrophils and probably macrophages as well generate superoxide anion as a primary product of the respiratory burst upon stimulation. Other active oxygen species including hydrogen peroxide seem to be formed within phagosomes. (3) A special cytochrome present in the membrane of phagocytes is probably involved in superoxide production.

We assessed the generation of reactive oxygen species (ROS: O2-, H2O2, OH . , chemiluminescence) by neutrophils and monocytes from six patients with infectious mononucleosis, ten patients with other viral diseases, and ten normal controls. Neutrophils from infectious mononucleosis patients showed markedly decreased generation of all reactive oxygen species, compared with the two control groups; this abnormality persisted for four to eight weeks after disease onset. Monocytes from these patients generated normal levels of ROS. Normal neutrophils incubated with T lymphocytes from infectious mononucleosis patients generated significantly less of each ROS than did those incubated with T cells from either control group. T cell-mediated suppression of ROS generation required both OKT4+ cells from infectious mononucleosis patients and OKT8+ cells from either patients or normals. We conclude that the generation of reaction oxygen species in neutrophils is suppressed in patients with infectious mononucleosis, at least in part, by interacting subsets of T lymphocytes.

We have examined whether the toxic effects of homocysteine on cultured endothelial cells could result from the formation and action of hydrogen peroxide. In initial experiments with a cell-free system, micromolar amounts of copper were found to catalyze an oxygen-dependent oxidation of homocysteine. The molar ratio of homocysteine oxidized to oxygen consumed was approximately 4.0, which suggests that oxygen was reduced to water. The addition of catalase, however, decreased oxygen consumption by nearly one-half, which suggests that H2O2 was formed during the reaction. Confirming this hypothesis, H2O2 formation was detected using the horseradish peroxidase-dependent oxidation of fluorescent scopoletin. Ceruloplasmin was also found to catalyze oxidation of homocysteine and generation of H2O2 in molar amounts equivalent to copper sulfate. Finally, homocysteine oxidation was catalyzed by normal human serum in a concentration-dependent manner. Using cultured human and bovine endothelial cells, we found that homocysteine plus copper could lyse the cells in a dose-dependent manner, an effect that was completely prevented by catalase. Homocystine plus copper was not toxic to the cells. Specific injury to endothelial cells was seen only after 4 h of incubation with homocysteine plus copper. Confirming the biochemical studies, ceruloplasmin was also found to be equivalent to Cu++ in its ability to cause injury to endothelial cells in the presence of homocysteine. Since elevated levels of homocysteine have been implicated in premature development of atherosclerosis, these findings may be relevant to the mechanism of some types of chronic vascular injury.