Increased serum catalase activity in rats subjected to thermal skin injury

We found that: rats subjected to thermal skin injury (burn) had increased serum hydrogen peroxide (H₂O₂) scavenging activity, serum catalase activity, erythrocyte (RBC) fragility, and edematous lung injury (lung leak) when compared to sham-treated rats. Serum H₂O₂ scavenging activity was inhibited by addition of sodium azide, a catalase inhibitor. Treatment of rats with the oxygen radical scavenger, dimethylthiourea (DMTU), decreased RBC fragility and lung leak but did not alter increased H₂O₂ scavenging or catalase activity of serum from rats subjected to skin burn. We conclude that increased serum catalase activity is a consequence of thermal skin injury and that increased serum catalase activity may be a mechanism that modulates H₂O₂-dependcnt processes following skin burn.All work was done during Dr. Leff's tenure of a Clinician-Scientist Award from the American Heart Association and supported by grants from the National Institutes of Health (PO1-AM-35098, BRS 983), Colorado Heart Association, American Lung Association, Ronald McDonald Children's Charities, Johnson & Johnson, Williams Family Foundation, Council for Tobacco Research Inc., Swan, Hill, Kleberg, Sachs, Etonner and American Express Foundations.     

Stimulation of human neutrophilic granulocytes by two monocytederived cytokines

Human monocytes upon stimulation with bacterial lipopolysaccharide release two cytokines which modulate the functions of neutrophilic granulocytes (PMN), a monocyte-derived chemotaxic (MOC) and tumor necrosis factor (TNF). Both cytokines stimulated the adherence of PMN on protein-coated nylon-fibers. Whereas MOC is one of the four most potent chemoattractants known, TNF was a most powerful inhibitor of PMN chemotactic migration towards several chemotactic factors including MOC. Neither cytokine stimulated the release of superoxide anion (O ₂ ^– ) or hydrogen peroxide (H₂O₂) from PMN in suspension. However, TNF, but not MOC, caused the release of considerable amounts of H₂O₂ and O ₂ ^– from PMN attached to nylon fibers. The two cytokines have similar effects on the adherence, opposing effects on chemotactic migration and different effects on the oxidative burst of PMN.     

Occupancy of adenosine receptors on human neutrophils inhibits respiratory burst stimulated by ingestion of complement-coated particles and occupancy of chemoattractant but not Fc receptors

Chemoattractants are generated at inflammatory loci that not only induce neutrophils (PMNs) to leave the vasculature but also stimulate PMNs to release potentially toxic agents (e.g., H₂O₂, O ₂ ^– or OH). We have recently demonstrated that endothelium releases adenosine which, when bound to a specific receptor on the PMN surface, inhibits release of toxic oxygen metabolites from stimulated PMN. To determine whether occupancy of adenosine receptors modulates generation and release of oxygen metabolites, we have studied the effect of 2-chloroadenosine on O ₂ ^– generation and O₂ consumption in response to opsonized zymosan particles (STZ) and immune complexes (IC). 2-Chloroadenosine inhibits, in a dose-dependent fashion, Of generation by neutrophils that have been exposed to C3b-coated particles (STZ). Inhibition of Of generation is similar in the presence or absence of cytochalasin B (IC₅₀=53 ±19 and 16 ±5nM, respectively,P=NS). Since occupancy of adenosine receptors might inhibit only externalization but not generation of oxygen metabolites, we studied the effect of 2-chloroadenosine on oxygen consumption by activated neutrophils. 2-Chloroadenosine inhibited O₂ consumption stimulated by STZ and the surrogate bacterial chemoattractant FMLP; however, inhibition of O₂ consumption varied with the presence or absence of cytochalasin B. In contrast, when neutrophils were stimulated by immune complexes, 2-chloroadenosine only minimally inhibited O ₂ ^– release and O₂ consumption (10 ± 5 and 5 ± 4% inhibition, respectively). Thus, occupancy of adenosine receptors inhibits O₂ consumption in parallel with inhibition of O ₂ ^– release. These results support the hypothesis that ingestion of complement-opsonized particles stimulates the respiratory burst by a mechanism different from that by which the respiratory burst is stimulated after occupancy of Fc receptors. Moreover, these observations suggest that endothelium, by releasing adenosine, prevent activated neutrophils from damaging the microvasculature at inflammatory loci. In contrast, deposition of immune complexes in vessel walls leads to vascular damage because endothelial cells are incapable of preventing attack by immune complex-stimulated neutrophils.This research was supported by grants from the U.S. Public Health Service (AI-10343 and HL29034) and the Veterans Administration.Dr. Cronstein is the recipient of a Clinical Investigator Award (K11-AR-01490) and was a fellow of the Arthritis Foundation. Dr. Broekman was an Established Investigator of the American Heart Association.     

PHA- and con A-induced chemiluminescence of human blood mononuclear cells and granulocytes in luminol or lucigenin

Phytohemagglutinin (PHA) and concanavaiin A (Con A) can evoke a chemiluminescence (CL) response both in granulocytes and blood mononuclear cells. We have used two parallel systems to compare the quantity and quality of oxygen radicals produced during activation. While the luminol-enhanced CL response is linked to the myeloperoxidase-H₂O₂-chloride system, the lucigenin-dependent light production measures only the Superoxide radical. PHA produced higher CL response in the presence of luminol than with lucigenin. Con A showed high CL response only in the lucigenin-enhanced system. The results suggest that while Con A induces mostly Superoxide production, the membrane stimulus evoked by PHA produces light through the myeloperoxidase-H₂O₂-halide system. Granulocytes are less sensitive to PHA than the blood mononuclear cells. The sensitivity of the responses to several scavengers and enzymes support the differential production of oxygen radicals following activation via these two lectins.     

Comparison of the effects of antioxidant non-steroidal anti-inflammatory drugs against myeloperoxidase and hypochlorous acid luminol-enhanced chemiluminescence

The interaction of myeloperoxidase (MPO) with H₂O₂ and Cl^– provides a potent antimicrobial/cytotoxic system for polymorphonuclear leukocytes (PMNs). MPO-related cytotoxicity may be associated with the formation of toxic oxidant MPO intermediates, HOCl, or both. MPO itself is able to oxidize drugs and cellular components. Non-steroidal anti-inflammatory drugs (NSAIDs) able to act as antioxidant free radical scavengers have recently been shown to inhibit luminol-enhanced chemiluminescence (CL) which results from the MPO–H₂O₂–Cl^– reaction. CL is a measure of the activity of this reaction. At that time it was not clear whether the source of CL which these NSAIDs affected was HOCl or components of the initial MPO–H₂O₂–Cl^– reaction. A NSAID antioxidant mechanism could affect MPO oxidant intermediates and HOCl.This study compares the effects of antioxidant NSAIDs, methylprednisone and free radical scavengers against MPO-based and NaOCl-based luminol-enhanced CL. Most NSAIDs which affected both MPO and NaOCl-CL appeared to share similar mechanisms, suggesting that MPO oxidant internediates and HOCl are susceptible to NSAID effects. However, most NSAIDs were more effective against MPO-CL. The effect of these NSAIDs against MPO-CL followed the profile of NSAIDs effective in previous studies against PMN-CL. One exception to this was methylprednisone, which has no effect on PMN or MPO-CL, yet inhibited NaOCl-CL. This and other data suggest that MPO and not HOCl-related reactions are a major source of PMN-CL. Less effective NSAIDs affected NaOCl-CL better than MPO-CL. While both HOCl and MPO oxidant intermediates may be affected by NSAIDs, it appears that MPO oxidant intermediates or MPO itself are the primary target for NSAID antioxidant free radical scavenging mechanisms. These antioxidant effects impair the major killing system of the PMN and may be NSAIDs' primary anti-inflammatory mechanism. Although our data suggests the production of superoxide anion and hydroxyl radical from the MPO–H₂O₂–Cl^– reaction, the actual presence or involvement of these free radical species is not confirmed herein.     

Tumor cell lysis by activated human neutrophils: Analysis of neutrophil-delivered oxidative attack and role of leukocyte function-associated antigen 1

The lysis of tumor cells, and other nucleated mammalian cells, by neutrophilic polymorphonuclear leukocytes (PMNs) triggered by phorbol myristate acetate (PMA) represents a widely used model system to dissect the PMN cytolytic armamentarium, potentially responsible for the cell damage at tissue sites of PMN activation. Although oxidants are generally considered to be instrumental in the target lysis by PMNs, the mediators actually involved remain a matter of controversy. Moreover, other factors potentially crucial to the lysis have not been clearly identified. In order to reexamine the determinants of the cytolytic process, we studied the events underlying the PMA-triggered PMN-delivered attack against two different targets, selected on the basis of preliminary experiments (B lymphoblastoid Daudi cells and erythroleukemic K 562 cells). The results suggest that the lysis is promoted by hypochlorous acid (HOCl) or a compound with characteristics very similar to HOCl itself. No evidence was obtained for the intervention or contribution of hydrogen peroxide (H₂O₂), hydroxyl (OH ·) radicals, and the major HOCl-derived chloramines. PMNs appeared to use 35% of the generated H₂O₂ to produce HOCl, while the remainder appears to be consumed by PMNs themselves and target cells as well. Moreover, PMNs and target cells coaggregated at an early step of the cytolytic reaction, through a process efficiently prevented by a monoclonal antibody (MoAb J-90) directed against leukocyte function-associated antigen-1 (LFA-1). The inhibition of the PMN-target aggregation by the MoAb J-90 resulted in the impairment of the lysis, despite a normal generation of HOCl. Thus, the data demonstrate that the PMA-triggered lysis of tumor target cells by PMNs requires at least two events, occurring simultaneously: the LFA-1-mediated effector-target adherence and the PMN production of HOCl. The intervention of the LFA-1-mediated PMN-target adherence in the PMA-triggered lysis is likely to allow PMNs to focus HOCl on the target cell surface and suggests that the process requires a sort of molecule to molecule recognition at the effector-target surface level.Supported by grants from Italian C.N.R. and M.P.I. Dr. Luciano Ottonello is a recepient of a Fellowship from C.N.R.     

Differential generation of chemiluminescence — detectable oxygen radicals by normal polymorphonuclear leukocytes challenged with sera from systemic lupus erythematosus and rheumatoid arthritis patients

Summary The generation of chemiluminescence (CL)-detectable oxygen radicals by normal human polymorphonuclear leukocytes (PMN) after challenging with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) sera is described. CL was measured in a luminol-dependent assay and referred to a standard obtained when preformed immune complexes (Ic) (human tetanus toxoid-antitoxoid Ic resuspended in normal pooled serum) were tested on PMN. Normal sera gave rise to CL activity by PMN between 0% and 50% of the standard Ic (mean±standard error of the mean (SEM): 20.7±4.8). Sera from SLE and RA patients induced strikingly different biological effects on PMN. SLE sera generally induced a high CL-detectable generation of oxygen metabolites which may be causally related to the intense tissue damage (vasculitis) frequently observed in this disease. In contrast to SLE, RA sera induced a CL-detectable respiratory burst by PMN that was included in the normal range. Thus, the biological effects of these sera in terms of stimulation of toxic oxygen radical generation by phagocytes are quite different. This generation of oxygen radicals might reflect a different clearance of circulating Ic by PMN in SLE and RA disease.Abbreviations CL Chemiluminescence - PMN Polymorphonuclear Leukocytes - SLE Systemic Lupus Erythematosus - RA Rheumatoid Arthritis - Ic immune complexes - TAT tetanus-antitetanus toxoid - TAT-S tetanus-antitetanus toxoid resuspended in pooled, non-inactivated human serum - SEM standard error for the mean - EHM Eagle-Hepes-Medium - PBS phosphate-buffered-saline - RF rheumatoid factor - SPA staphylococcal protein A - ANA antinuclear antibodies - KD kilodaltons - Ag antigen     

Enhancement of polymorphonuclear leukocyte (PMN) function by OK-432

The influence of OK-432, a streptococcal preparation, on human polymorphonuclear leukocytes (PMN) was examined. OK-432 increased O₂⁻ generation by PMN in oral cancer patients, and sustained the production of O₂⁻ in patients on chemoradiotherapy. Enhanced O₂⁻ generation was also observed when PMN were cultured with 10⁻² KE/ml OK-432 for 1 h and then stimulated with phorbol myristate acetate or formyl-metionyl-leucil-phenylalanine (FMLP). In addition, PMN O₂⁻ generation was promoted by culture supernatants of peripheral blood mononuclear cells (PBMC) incubated with 10⁻³ or 10⁻² KE/ml OK-432. Furthermore, OK-432 (10⁻³−10⁻² KE/ml) enhanced the chemiluminescence of FMLP- and PMA-stimulated PMN. However, nitroblue tetrazolium reduction and myeloperoxidase activity were only minimally enhanced. Not only the candidacidal activity of PMN but also antibody-dependent cell-mediated cytotoxicity against Candida and Raji cells were enhanced in correspondence with the increased generation of reactive oxygen species.Culture of PMN or PBMC for 24 h with OK-432 resulted in a concentration-dependent increase in the substantial production of interleukin-1β, interleukin-6 and tumor necrosis factor-α. OK-432 also enhanced granulocyte-macrophage colony stimulating factor and gamma-interferon generation by leukocytes in a dose-dependent manner. Our research indicates that OK-432 enhances PMN function directly as well as via the promotion of cytokine production, and suggests that these effects of OK-432 could be beneficial in immunosuppressed patients.     

Antioxidation theory of non-steroidal anti-inflammatory drugs based upon the inhibition of luminol-enhanced chemiluminescence from the myeloperoxidase reaction

The action of non-steroidal anti-inflammatory drugs (NSAIDS) has been ascribed to their ability to block the reaction of arachidonate with cyclooxygenase/peroxidase, thus inhibiting the cellular production of inflammation mediators such as prostaglandins and leukotrienes. However, this and other polymorphonuclear leukocyte (PMN) peroxidases such as myeloperoxidase (MPO) would still be capable of producing destructive oxidants which contribute to inflammation. Sulindac sulfide (Clinoril^® sulfide) has recently been shown to scavenge oxidant products of prostaglandin cyclooxygenase/peroxidase and MPO. The MPO–H₂O₂–Cl^– reaction is a potent antimicrobial/cytotoxic system which produces HOCl, a strong oxidant. MPO itself has the ability to oxidize drugs and cellular components, and may be the main oxidant in PMN defenses. An antioxidant/free radical scavenger action of NSAIDs against the MPO system could be a primary mechanism of their anti-inflammatory effects. Other antioxidant/free radical scavengers have anti-inflammatory effects.MPO activity has previously been quantified using chemiluminescence (CL). In this study, NSAIDs from various classes were tested for their ability to inhibit luminol-enhanced CL from MPO. The most potent NSAIDs against MPO-CL were BW755C, phenylbutazone, indomethacin and sulindac sulfide. Salicylates and arylacetic acid derivatives, such as naproxen, also decreased MPO-CL. These drugs are also effective against CL from PMNs, of which MPO may be a main source. This effect of NSAIDs on MPO suggests that NSAIDs may impair the killing mechanism of the PMN, preventing cell destruction and release of inflammation mediators. PMN MPO appears to be a target for the antioxidant/free radical scavenging effects of NSAIDs.     

Altered function of synovial fluid granulocytes in patients with acute inflammatory arthritis

In rheumatoid arthritis (RA) a chronic inflammatory state exists in which the synovial fluid is periodically filled with large numbers of polymorphonuclear leukocytes (PMNs). Oxygen radicals produced by these cells have been implicated as mediators of tissue damage and may be directly involved in the pathogenesis of RA. We examined the production of oxygen radicals by synovial fluid PMNs (SFPMNs) and peripheral blood PMNs (PB-PMNs) by measuring chemiluminescence (CL) as well as Superoxide anion (O ₂ ^– ) release. Increased spontaneous CL in the presence of luminol and increased CL in response to phorbol myristate acetate (PMA) was observed in SF-PMNs when compared to PB-PMNs. When zymosan was used as the stimulus in the absence of luminol, a slightly lower CL response was observed in SF-PMNs as compared to PB-PMNs. No significant differences were observed in the generation of O ₂ ^– generation with any stimulus. Preincubation of normal PBPMNs in 10% synovial fluid enhanced the luminol-dependent spontaneous and PMA-stimulated CL as well as zymosan-stimulated CL. When O ₂ ^– release from normal PB-PMNs pretreated with 10% synovial fluid was compared to untreated controls, enhancement of spontaneous O ₂ ^– release was observed. PMA- and zymosan-stimulated responses did not differ significantly from controls. Increased spontaneous and PMA-stimulated release of myeloperoxidase (MPO) was also observed in normal PB-PMNs pretreated with synovial fluid. These findings may explain the increased luminol-dependent CL since this type of CL requires the presence of MPO. Our findings suggest that the enhanced chemiluminescence observed in normal PMNs treated with synovial fluids may be related to increases in spontaneous O ₂ ^– generation and myeloperoxidase release. Increased MPO release may account for enhanced CL observed in SF-PMNs.     

The influence of chemotactic factors on neutrophil adhesiveness

The ability of several chemotactic factors to alter polymorphonuclear neutrophil (PMN) adhesiveness to nylon fibers was studied. Partly purified bacterial chemotactic factor, the isolated chemotactic fragment of human C5, and the chemotactic synthetic tripeptide, formyl-methionyl-leucylphenylalanine, transiently enhanced the nylon fiber adhesiveness of rabbit peritoneal PMNs. The capacity of these chemotactic factors to augment PMN adherence closely paralleled their ability to aggregate PMNs in suspension and to induce neutropenia when infused into rabbits. However, at least a portion of the adherence-augmenting capacity of these agents was independent of their ability to induce PMN aggregation. Thus, chemotactic factors appear to transiently enhance PMN adhesiveness to a variety of surfaces. This hyperadhesiveness may underlie the augmented nylon fiber adherence, aggregation, and neutropenia induced by these factors.Research supported in part by NIH Grants AI-113910, HL-07202, and HL-05474.     

Synergy between tumour necrosis factor α and granulocyte-macrophage colony-stimulating factor in neutrophil stimulation

We have examined the interaction between the cytokines, granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumour necrosis factor α (TNFα) on polymorphonuclear leukocyte (PMN) function and on PMN responses to further stimulation with formyl Met Leu Phe (fMLP). Incubation of PMN with TNFα (0.3 nM) and, to a lesser extent, with GM-CSF (1 nM) directly stimulated superoxide anion (O ⁻₂ ) generation and increased the response to subsequent stimulation of PMN by fMLP (100 nM). However, the combination of GM-CSF and TNFα did not result in increased O ⁻₂ generation and there was no synergistic effect of the combination of these cytokines on the priming of fMLP-induced O ⁻₂ generation. The combination of TNFα and GM-CSF did result in a striking synergism in the stimulation of PAF generation, and, whereas neither stimulus alone resulted in detectable PAF release, the combination elicited the release of significant levels of PAF. The observation of significant PAF release from PMN exposed to TNFα and GM-CSF indicates that overt neutrophil stimulation with phagocytic or soluble stimuli may not be required for expression of at least some of the PMN pro-inflammatory capacity.

     

The effect of adherence on the generation of reactive oxygen species by human neutrophilic granulocytes

Human neutrophilic granulocytes (PMN) suspended in protein containing salt solution or adherent on protein coated nylon fibers were tested for the production of H₂O₂ and O ₂ ^– in response to various PMN stimulants. Upon stimulation with the chemotactic factors formyl-methionyl-leucyl-phenylalanine, C5a and platelet activating factor, the non-chemotactic ionophore A23187, and the chemotaxis inhibitors tumor necrosis factor (TNF) and lymphotoxin (TNF ) adherent PMN produced considerably more reactive oxygen metabolites than suspended cells. The relative amounts of the two metabolites varied with the stimulus and its concentration, TNF and TNF favoring H₂O₂ production, C5a eliciting more O ₂ ^– than H₂O₂ and the other active stimulants being in between. Leukotriene B₄ and a novel monocytederived chemotaxin were inactive in releasing either oxygen derivative from adherent or suspended PMN. The data indicate that attachment of PMN to endothelial cells or to connective tissue substances can strongly enhance its ability to respond to a given stimulus with the production of reactive oxygen metabolites. The findings may in part explain the priming phenomenon since many PMN-priming mediators increase the cells' adherence.     

In vitro effect of asbestos fibers on polymorphonuclear leukocyte function

Incubation of chrysotile and amphibole asbestos fibers with normal human peripheral blood polymorphonuclear leukocytes (PMN) resulted in a significant stimulation of PMN metabolic activity and generation of toxic oxygen by-products as measured by chemiluminescence (CL). Although all asbestos fibers tested were cytotoxic to PMN, cytotoxicity and CL varied disproportionately with fiber type. Anthophyllite asbestos produced the greatest PMN cytotoxicity. It also depressed PMN phagocytosis of latex beads the most and induced the greatest PMN CL response of the fiber types examined. We postulate that asbestos-induced release of toxic oxygen by-products from PMN which have infiltrated into the pulmonary alveoli may contribute to disease pathogenesis in asbestosis.     

Cytokine toxicity in insulin-producing cells is mediated by nitro-oxidative stress-induced hydroxyl radical formation in mitochondria

Although nitric oxide (NO) and oxidative stress both contribute to proinflammatory cytokine toxicity in pancreatic β-cells during type 1 diabetes mellitus (T1DM) development, the interactions between NO and reactive oxygen species (ROS) in cytokine-mediated β-cell death have not been clarified. Exposure of insulin-producing RINm5F cells to IL-1β generated NO, while exposure to a combination of IL-1β, TNF-α, and IFN-γ, which simulates T1DM conditions, generated both NO and ROS. In theory, two reactions between NO and ROS are possible, one with the superoxide radical yielding peroxynitrite, and the other with hydrogen peroxide (H₂O₂) yielding hydroxyl radicals. Results of the present work exclude peroxynitrite involvement in cytokine toxicity to β-cells because its generation did not correlate with the toxic action of cytokines. On the other hand, we show that H₂O₂, produced upon exposure of insulin-producing cell clones and primary rat islet cells to cytokines almost exclusively in the mitochondria, reacted in the presence of trace metal (Fe⁺⁺) with NO forming highly toxic hydroxyl radicals, thus explaining the severe toxicity that causes apoptotic β-cell death. Expression of the H₂O₂-inactivating enzyme catalase in mitochondria protected against cytokine toxicity by preventing hydroxyl radical formation. We therefore conclude that proinflammatory cytokine-mediated β-cell death is due to nitro-oxidative stress-mediated hydroxyl radical formation in the mitochondria.     

Luminol-independent chemiluminescence by phagocytes is markedly enhanced by dexamethasone,not by other glucocorticosteroids

The effect of several glucocorticosteroids on the generation of reactive oxygen species (ROS) was examined. The ROS assessed were O ₂ ^– , H₂O₂, OH·, and chemiluminescence (CL) (determined in the presence or absence of luminol), generated by both opsonized zymosan-stimulated neutrophils or monocytes and by the xanthine-xanthine oxidase system. Except for luminol-independent CL, only high concentrations (10^–4 M) of steroids could decrease each ROS. In contrast, luminol-independent CL generation in the phagocyte system was increased in a dose-dependent manner by the addition of dexamethasone, but not by any other steroid. Further, in lymphocyte cultures stimulated with Con A for four days, luminol-independent CL generation was demonstrated and enhanced by the addition of dexamethasone, although CL generation was not detected in the absence of dexamethasone. These findings provide evidence that CL does not always represent light specific to ROS, and they suggest the possibility that dexamethasone induces emission of light at sites of inflammation.     

Adherence and Migration of Guinea Pig Granulocytes After Enzyme Treatment of the Cell Surface

Glycogen-induced polymorphonuclear granulocytes (PMN) from the peritoneal cavity ofguinea pigs were examined (1) for their adherence to nylon fibers in the absence and presence of the adherence-enhancing chemotactic peptide formyl-methionyl-leucyl-phenylalanine (f-MLP), (2) for their random migration through the filter of a Boyden chamber and (3) for their chemotactic migration towards f-MLP. The cells were analyzed before and after treatment with the enzymes neuraminidase, papain and trypsin. PMN adhesiveness was increased by neuraminidase digestion but reduced by treatment with the proteolytic enzymes. Neuraminidase and trypsin had no effect on cell migration, while papain reduced random migration without affecting f-MLP-induced chemotaxis. The data suggest that the type of adherence measured by the nylon fiber method differs from the temporary attachment of cells migrating through a chemotaxis filter towards an attracting substance.     

Chemiluminescence in activated human neutrophils

Human neutrophils (PMNs) suspended in Hanks' balanced salt solution (HBSS), which are stimulated either by polycation-opsonized streptococci or by phorbol myristate acetate (PMA), generate nonamplified (CL), luminol-dependent (LDCL), and lucigenin-dependent chemiluminescence (LUCDCL). Treatment of activated PMNs with azide yielded a very intense CL response, but only a small LDCL or LUCDCL responses, when horse radish peroxidase (HRP) was added. Both CL and LDCL depend on the generation of Superoxide and on myeloperoxidase (MPO). Treatment of PMNs with azide followed either by dimethylthiourea (DMTU), deferoxamine, EDTA, or detapac generated very little CL upon addition of HRP, suggesting that CL is the: result of the interaction among H₂O₂, a peroxidase, and trace metals. In a cell-free system practically no CL was generated when H₂O₂ was mixed with HRP in distilled water (DW). On the other hand significant CL was generated when either HBSS or RPMI media was employed. In both cases CL was markedly depressed either by deferoxamine or by EDTA, suggesting that these media might be contaminated by trace metals, which catalyzed a Fenton-driven reaction. Both HEPES and Tris buffers, when added to DW, failed to support significant HRP-induced CL. Nitrilotriacetate (NTA) chelates of Mn²⁺, Fe²⁺, Cu²⁺, and Co²⁺ very markedly enhanced CL induced by mixtures of H₂O₂ and HRP when distilled water was the supporting medium. Both HEPES and Tris buffer when added to DW strongly quenced NTA-metal-catalyzed CL. None of the NTA-metal chelates could boost CL generation by activated PMNs, because the salts in HBSS and RPMI interfered with the activity of the added metals. CL and LDCL of activated PMNs was enhanced by aminotriazole, but strongly inhibited by diphenylene iodonium (an inhibitor of NADPH oxidase) by azide, sodium cyanide (CN), cimetidine, histidine, benzoate, DMTU and moderately by Superoxide dismutase (SOD) and by deferoxamine. LUCDCL was markedly inhibited only by SOD but was boosted by CN. Taken together, it is suggested that CL generated by stimulated PMNs might be the result of the interactions among, NADPH oxidase, (inhibitable by diphenylene iodonium), MPO (inhibitable by sodium azide), H₂O₂ probably of intracellular origin (inhibitable by DMTU but not by catalase), and trace metals that contaminate salt solutions. The nature of the salt solutions employed to measure CL in activated PMNs is critical.     

Effects on extra- and intracellularly localized,chemoattractant-induced,oxygen radical production in neutrophils following modulation of conditions for ligand-receptor interaction

Results obtained with the luminol-dependent chemiluminescence (CL) technique show that with this technique, generation of oxygen radicals from an extra-as well as from an intracellular source is quantified. This investigation was performed in order to study the relationship between intra and extracellularly generated radicals in human granulocytes stimulated with the chemoattractant formyl-methionyl-leucyl-phenylalanine (FMLP). A difference in time course between extra and intracellular CL was observed. The extracellular response reached a maximum value after 1–2 min, whereas the intracellular response reached a maximum value after 5–7 min. The ED₅₀ values for the two responses were the same, whereas the onset time was a little longer for the intracellular response. Both high and low concentrations of FMLP gave rise to CL. However, the ratio between the extra and the intracellular response differed depending on the concentration of FMLP; the ratio was decreased at low concentrations of FMLP. The same type of change was obtained when the ligand-receptor ratio was decreased through modulation of the number of exposed receptors. The ratio between extra and intracellular activities was also changed by cytochalasin B, removal of Ca²⁺, or removal of Na⁺. The role of the extra and intracellular oxygen radical production as well as possible regulatory mechanisms are discussed.