Subcellular localization of H2O2 production in human neutrophils stimulated with particles and an effect of cytochalasin-B on the cells

The ultrastructural localization of H2O2 production in suspended polymorphonuclear leukocytes (PMN) stimulated with particles was studied using CeCl3 technique. PMN stimulated with opsonized zymosan or polystylene latex with or without IgG were incubated in 0.1 M Tris- maleate buffer with 1 mM CeCl3 and 10 mM aminotriazole. Cells were then fixed and embedded in a resin for electron microscopy. The reaction product of cerium perhydroxide was observed on the phagosomal membranes and on the areas of the plasma membrane engulfing the particles. Catalase or ferricytochrome-c decreased the deposits. p-Benzoquinone (O2- scavenger) inhibited the formation of the deposits, but KCN or NaN3 enhanced it. Pretreatment with p-diazobenzenesulfonic acid inhibited the reaction. In some PMN pretreated with cytochalasin-B, cellular aggregation was observed. The H2O2 production in these cells were observed on the membrane adherent to the particles and on the contact surface of the membrane of adjoining PMN. The plasma membrane was damaged and the electron-dense product was diffused into the cytoplasm. These results clearly show that H2O2 production is initiated at the area of the plasma membrane adherent to the particles and that H2O2 is released before the completion of phagocytosis.     

Inhibition of zymosan activation of human neutrophil oxidative metabolism by a mouse monoclonal antibody

We have studied a neutrophil-specific murine monoclonal antibody, PMN7C3 (IgG3), which specifically alters PMN oxidative metabolism stimulated by serum-opsonized zymosan (STZ) or Candida albicans (STC). Polymorphonuclear cells (PMNs) exposed to PMN7C3 show a significant depression in O2- release (52.8% +/- 2.5% of control), H2O2 release (44.4% +/- 6.0% of control), and O2 consumption (73.9% +/- 2.6% of control) in response to STZ. O2 release in response to phorbol myristate acetate (PMA) was modestly reduced (78.4% +/- 3.7%) by PMN7C3 treatment, but not to the extent seen with STZ or STC. PMN7C3 did not affect O2 release by PMNs stimulated by zymosan opsonized with IgG or by S. aureus, A 23187, or FMLP. PMN7C3 was not cytotoxic, did not trigger oxidative metabolism when used as a stimulus, did not alter STZ- induced degranulation, and did not interfere with binding or uptake of STZ by PMNs. Exposure of PMNs to PMN7C3 decreased PMN rosette formation with erythrocytes coated with C3b (54% of control) or C3bi (63% of control), but had no affect on rosette formation with IgG-coated erythrocytes. PMN7C3 does not bind to monocytes and had no affect on rosette formation by this cell type. Binding of antibody PMN7C3 to the neutrophil surface inhibits the oxidative response to opsonized STZ or STC, possibly in part by altering the function or expression of C3b and C3bi receptors. Monoclonal antibodies such as PMN7C3 provide highly specific probes that may be used to define the molecular features of the stimulus-coupled response of PMN activation.     

Modulation of polymorphonuclear leukocyte function by cetiedil

Cetiedil citrate monohydrate inhibits sickling of red cells and aggregation of platelets. We assessed its ability to attenuate polymorphonuclear leukocyte (PMN) function. PMN aggregation in response to 2 X 10(-7) M formyl-met-leu-phe (FMLP) was inhibited in a dose- dependent fashion by cetiedil concentrations ranging from 60 to 250 microM. Additionally, 125 microM cetiedil inhibited PMN aggregation in response to 2 X 10(-7) M FMLP, 20 ng/ml phorbol myristate acetate (PMA), and 1 X 10(-6) M A23187 by 69% +/- 18%, 72% +/- 20%, and 65% +/- 4%, respectively. Inhibition of FMLP-induced aggregation was provided by only 5 min of incubation of the drug with the cells and was partially reversible. Cell viability was unaffected by exposure of PMN to the drug. Correspondingly, 125 microM cetiedil prevented the translocation of calcium from the PMN membrane as assessed by chlorotetracycline fluorescence. Paralleling the effect of the drug on PMN aggregation, 125 microM cetiedil inhibited release of superoxide by 55% and decreased the number of available 3H-FMLP receptors. However, its effect on release of the primary granule constituent, myeloperoxidase, was minimal (4.5% inhibition), while the effect on release of the specific granule product, lactoferrin (27% inhibition), was modest. These studies indicate that cetiedil affects PMN aggregation and superoxide release to a much greater extent than PMN degranulation. Thus, cetiedil may have potential uses in modulating inflammatory response in vivo.     

Assessment of neutrophil aggregation by Coulter® STKR and STKS haematological analysers

Summary. We have studied an alternative method to aggregometry for the assessment of human polymorphonuclear (PMN) leucocyte aggregation. This simple, rapid and reliable procedure counts unaggregated cells on both Coulter’STKS and STKR haematological analysers by the impedance principle. Aggregation of PMN was induced by 15 min incubation with fresh autologous serum (FAS) after a 10 min phorbol myristate acetate (PMA) activation of neutrophils in small aliquots (0.25 ml) of suspension containing about 4.0 × 10⁹ PMN/l. Differences (x 100) between count of resting and PMA + FAS treated neutrophils/count of resting PMN reflect percent aggregation. By this procedure, PMN aggregation did not occur in autologous plasma from EDTA anticoagulated whole blood; it was partially inhibited by hydrocortisone, whereas inactivated or Zymosan activated sera gave values similar to those from FAS induced aggregation. PMA aggregation was dependent on Ca²⁺+Mg²⁺ concentration. Intra-assay analytical variability did not exceed 4% on either instrument. Reference values (n= 20) of percent PMN aggregation were 50.7 ± 4.7 on STKS and 47.1 ± 4.8 on STKR. Most probably, the interindividual variance was due to the physiological variability of Mg²⁺ and/or Ca²⁺ concentrations in FAS. Thus, this procedure reflects the true PMN aggregability status in a given subject, and in a given electrolyte environment.     

Volumetric changes in phorbol myristate acetate activated neutrophils: a rapid and simple assay using Coulter counter STKR and STKS hematological analyzers.

BACKGROUND: The aim of this study was to investigate some physical changes (volume, conductivity and scatter) in human polymorphonuclear leukocytes (PMN) activated with phorbol myristate acetate (PMA). METHODS: Volume changes in PMA-activated neutrophils were assayed by both STKR and STKS, two Coulter hematological analyzers. Scatter changes in PMN activated by PMA in suspensions containing nitro-blue tetrazolium (NBT) were investigated on STKS scattergrams. RESULTS: PMA activation induced PMN volumetric increases that could be assayed with STKR and displayed with STKS. The activation of PMN in suspension containing NBT induced cellular scatter changes on STKS scattergrams. The differences in scatter between resting and PMA-activated neutrophils may thus provide a semiquantitative assay of NBT reduction and superoxide production. Volume changes in PMA-activated neutrophils were due to cellular swelling through water uptake, induced by Na+/H+ antiport activation and Na+ influx. Since volume changes in PMA-activated neutrophils might occur without O2- production and vice versa, in the cases in which PMA activation of protein kinase C cannot be demonstrated by O2- production, the effect of PMA on protein kinase C- mediated Na+/H+ antiport activation, and in turn on PMA volume changes, reflects protein kinase C activation. CONCLUSIONS: A screening of neutrophils unresponsive to volumetric changes from PMA activation may be easily performed using both Coulter STKR and STKS analyzers, whereas expected or defective PMA-induced production of O2- may be semi-quantitatively evaluated by STKS.     

Protease inhibitor eglin-c affects superoxide anion release but not bacterial killing by human polymorphonuclear leukocytes

In order to assess the influence of the protease inhibitor eglin-c on superoxide anion (O-2) release by human polymorphonuclear leukocytes (PMN), cells were secured from normal donors and stimulated with phorbol myristate acetate (PMA), opsonized zymosan, or n-formyl-methionyl-leucyl-phenylalanine (FMLP). In the presence of 100 micrograms/ml eglin-c, the activation time was prolonged and the maximum linear rate of O-2 formation was depressed following stimulation with PMA; a concentration of 1000 micrograms/ml eglin-c was required to produce a similar effect with opsonized zymosan. Eglin-c did not influence the activation time following stimulation with FMLP, but at 2000 micrograms/ml, the protease inhibitor attenuated the rate of O-2 production in response to the chemotactic peptide. In the presence of cytochalasin B, the inhibitory effect of eglin-c on O-2 release following stimulation with FMLP became more pronounced. In spite of these alterations in O-2 formation, the protease inhibitor did not impair the bactericidal activity of PMN against Staphylococcus aureus. Therefore, we conclude that although eglin-c can disrupt the activation and the activity of the superoxide-generating system of human PMN, the effect is stimulus dependent and is not associated with an alteration in the microbicidal capacity of neutrophils against S. aureus.     

Metabolic, membrane, and functional responses of human polymorphonuclear leukocytes to platelet-activating factor

The phospholipid mediator of anaphylaxis, platelet-activating factor (PAF) is chemotactic for polymorphonuclear leukocytes (PMN). We have examined this agent's effects on several other PMN functions. Human PMN were prepared from heparinized venous blood by Ficoll gradient. Metabolic burst was examined by measurement of O2 use and O2.- production in the presence or absence of PAF (10(-6)--10(-9) M). Unless cells were treated with cytochalasin-B (5 micrograms/ml), no significant respiratory burst was demonstrated. However, pretreatment with PAF (10(-7) M) enhanced approximately threefold the O2 utilization found when cells were subsequently stimulated with 10(-7) M FMLP. PAF also stimulated arachidonic acid metabolism in 14C-arachidonic acid- labeled PMN. Thin-layer chromatography analysis of chloroform-methanol extracts showed substances that comigrated with authentic 5- hydroxyeicosatetraenoic acid had a marked increase in radioactivity following PAF stimulation at 10(-7) M. PAF failed to stimulate release of granule enzymes, B-glucuronidase, lysozyme, or myeloperoxidase unless cytochalasin-B were added. PAF from 10(-6) M to 10(-10) M affected PMN surface responses. PMN labeled with the fluorescent dye, chlorotetracycline, showed decreased fluorescence upon addition of PAF, suggesting translocation of membrane-bound cations. Further, the rate of migration of PMN in an electric field was decreased following PAF exposure, a change consistent with reduced cell surface charge. PMN self-aggregation and adherence to endothelial cells were both influenced by PAF (10(-6) M--10(-9) M). Aggregation was markedly stimulated by the compound, and the percent PMN adhering to endothelial cell monolayers increased almost twofold in the presence of 10(-8) M PAF. Thus, PAF promotes a variety of PMN responses: enhances respiratory burst, stimulates arachidonic acid turnover, alters cell membrane cation content and surface charge, and promotes PMN self- aggregation as well as adherence to endothelial cells.     

Lactoferrin: a promoter of polymorphonuclear leukocyte adhesiveness

Polymorphonuclear leukocytes (PMN) degranulate, adhere to vascular endothelium, or aggregate to each other following exposure of the cells to high concentrations of chemotactic stimuli such as formyl-methionyl- leucyl phenylalanine (FMLP). PMN released the specific granule product lactoferrin more readily in response to chemotactic stimuli, which correlated with promotion of PMN aggregation as measured by light transmission and enhanced PMN adherence. Both concanavalin A (Con-A) and phorbol myristate acetate (PMA), agents that lead to specific granule discharge, induced and sustained human PMN aggregation. Similarly, supernatants, generated from Con-A-treated PMN, aggregated fresh PMN in the presence of alpha-methylmannoside, a competitive inhibitor of the lectin. Anti-human lactoferrin IgG but not normal goat IgG blunted the aggregation elicited by both PMA and FMLP. Both human milk lactoferrin and rabbit PMN lactoferrin aggregated human lactoferrin promoted PMN adherence to endothelial cells. The enhanced PMN stickiness was correlated with the early phase of degranulation. Thus, PMN lactoferrin serves an autoregulatory role to retain PMN at inflammatory sites to amplify the inflammatory response.     

Enhancement of chemotactic factor-stimulated neutrophil oxidative metabolism by leukotriene B4

Leukotriene B4 (LTB4) is a potent primary stimulator of neutrophil chemotaxis, aggregation, and degranulation and induces superoxide production at higher concentrations. In order to determine whether LTB4 modulates neutrophil responses to oxidative stimuli, human neutrophils (PMNs) were incubated with LTB4 prior to stimulation with f-Met-Leu-Phe (fMLP, 10(-7) mol/L), opsonized zymosan (OZ, 250 micrograms/mL), or phorbol myristate acetate (PMA, 32 nmol/L). Superoxide (O2-) production by stimulated PMNs was assessed by the superoxide dismutase-inhibitable reduction of cytochrome c. LTB4 alone did not stimulate O2- production in concentrations below 10(-7) mol/L and had no effect on the O2- assay. In the concentration range of 10(-12) to 10(-8) mol/L, LTB4 did not alter O2- release induced by OZ or PMA. In contrast, LTB4-treated cells demonstrated enhanced O2- production following exposure to fMLP, and in the presence of 10 nmol/LLTB4, generated 180% +/- 41% of O-2 quantities produced by control cells (n = 23). Enhancement was LTB4 dose-dependent, was maximal in the range of 1 to 10 nmol/L LTB4, was not reversed by removal of the lipid from the medium prior to fMLP stimulation, and was not dependent on the presence of Ca++ or Mg++ in the suspending medium. Chemiluminescence of fMLP-stimulated neutrophils was increased to 323% of controls in neutrophils preincubated with 10 nmol/L LTB4. Unlike augmentation of oxidative responses to fMLP seen with other degranulating stimuli, enhancement by LTB4 was not correlated with an increase in 3H-fMLP receptor binding. These results indicate that, in addition to its primary effects on neutrophil function, LTB4 modulates PMN oxidative responses to the chemotactic peptide and, thus, may amplify the release of oxygen metabolites at inflammatory foci.     

The role of glutathione reductase in maintaining human granulocyte function and sensitivity to exogenous H2O2

Human granulocytes (polymorphonuclear leukocytes, PMN) produce H2O2 and other reactive oxygen species while undergoing phagocytosis. To examine the role of the glutathione cycle in metabolizing H2O2, we incubated PMN with 1,3-bis (2-chloroethyl) nitrosourea (BCNU). Incubation of PMN with BCNU results in a dose-dependent inhibition of PMN glutathione reductase (GRED), with 50% inhibition occurring at approximately 2 micrograms/mL BCNU. PMN hexose monophosphate shunt activity stimulated with an exogenous H2O2-generating system was inhibited only when the GRED activity was reduced to less than 30% of control. BCNU-treated cells contained lower levels of reduced sulfhydryls and reduced glutathione, which decreased even more in the presence of an exogenous H2O2-generating system. The effect of BCNU and exogenous H2O2 on various aspects of phagocytosis were examined. Exposure of BCNU-treated PMN to an H2O2-generating system resulted in an inhibition of chemotactic peptide-induced shape changes and degranulation. The ability of BCNU-treated cells to produce O2- was diminished only when the PMN were incubated with an H2O2-generating system in the presence of cyanide. Ingestion of opsonized bacteria by BCNU-treated PMN was unaffected by incubation in an H2O2-generating system even in the presence of cyanide. We conclude that PMN GRED is inhibited by BCNU, the ability of PMN to metabolize H2O2 is affected only when GRED is reduced more than 70%, this inhibition affects the glutathione content of these cells, and some, but not all of the phagocytic functions of GRED-inhibited PMN are inhibited after exposure to an H2O2-generating system.     

The effect of selected antiarrhythmic drugs on neutrophil free oxygen radicals production measured by chemiluminescence

Summary The evidence that free oxygen radicals produced by polymorphonuclear neutrophils (PMN) participate in generation of reperfusion arrhythmias is well documented. The in vitro effect of selected antiarrhythmic drugs on PMN free radicals production was evaluated by luminol- (LuCL) and lucigenin- (LgCL) amplified chemiluminescence stimulated with opsonized zymosan (o.z.) and phorbol myristate acetate (PMA). Fast sodium channel inhibitors varied in the influence on PMN chemiluminescence: from an inhibition in all models studied by procainamide, to lack of an effect by mexiletine. Propafenone, similarly to ajmaline and verapamil, inhibited LuCL stimulated with PMA, as well as LgCL after stimulation with both PMA and o.z. Bretylium tosylate decreased LuCL stimulated with both inducers, with no effect on LgCL. Amiodarone in high concentrations inhibited both LuCL and LgCL. -blockers propranolol and practolol impaired LuCL stimulated with o.z., as well as LgCL induced with PMA, whereas -blocker phentolamine inhibited LuCL and LgCL stimulated with both inducers. The drugs' effect on PMN free oxygen radicals production may constitute an additional mechanism of their activity.     

Hydrogen peroxide generation and its regulation in FRTL-5 and porcine thyroid cells.

Hydrogen peroxide acts as electron acceptor in the oxidative reactions (iodination and coupling) by which the thyroid hormones are formed. Regulation of the generation of H2O2 was studied in monolayer cultures of the FRTL-5 rat thyroid cell line and in primary monolayer cultures of porcine thyroid cells. Both cell types were grown in a medium containing either a six-hormone mixture, including TSH (6H), or a five-hormone mixture without TSH (5H) for 1 to several days before the experiment. The production of H2O2 was measured with the homovanillic acid fluorescence assay and expressed as picomoles of H2O2 formed per min/microgram DNA. In FRTL-5 cells grown in 6H medium, only a weak and varying stimulation of H2O2 production was induced by TSH at high concentration (greater than 10 mU/ml), and no stimulation was seen by TSH at low concentration or by 8-bromo-cAMP, whereas forskolin had a good stimulatory effect. In FRTL-5 cells grown in 5H medium for 1-3 days, all three substances were potent stimulators. In porcine thyrocytes examined in the same way, none of the three presumptive stimulators had any effect in 6H cultures, and only TSH (at high concentration) had a weak effect in 5H medium. ATP, a stimulator of the Ca2+/phosphatidylinositol cascade via a P2-purinergic receptor, had no effect on H2O2 generation in FRTL-5 cells in 6H medium. Phorbol myristate acetate (PMA), a direct activator of protein kinase-C, induced a weak stimulation in these cells. In FRTL-5 cells in 5H medium, both ATP and PMA evoked a strong, and similar, enhancement of H2O2 production. In porcine cells in 6H medium, ATP evoked a moderate and PMA a strong stimulation; the effects in 5H were similar to the corresponding effects in 6H medium. The observations are interpreted to show that in FRTL-5 cells the regulation of H2O2 generation uses both the cAMP cascade and the Ca2+/phosphatidylinositol cascade, whereas in porcine thyrocytes the cAMP route is unimportant. In FRTL-5 cells the Ca2+/phosphatidylinositol cascade may be influenced by the cAMP system.     

Con-A-stimulated superoxide production by granulocytes: reversible activation of NADPH oxidase

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.     

Modulation of human neutrophil effector functions by monoclonal antibodies against surface membrane molecules of 94,000 and 180,000 molecular weight

Function-related antigens on the neutrophil (PMN) surface were identified using two newly developed PMN-specific mouse monoclonal antibodies. These IgG antibodies, designated Ab 1-14 and Ab 1-15, were selected for detailed study after initial testing revealed their significant inhibition of PMN superoxide generation in response to N- formyl-Met-Leu-Phe (FMLP) (64% for 1-14 and 64% for 1-15; P less than .05). In further experiments, Ab 1-14 augmented PMN adhesion (by 111%; P less than .01) and degranulation (by 52%; P less than .05) in response to FMLP, while Ab 1-15 inhibited these responses by 42% and 29%, respectively (P less than .05). Ab 1-14 reduced PMN chemotaxis in response to FMLP by 37% (P less than .02), and unlike Ab 1-15, Ab 1-14 significantly reduced unstimulated PMN binding of complement-coated sheep red blood cells. Ab 1-14 and Ab 1-15 significantly reduced PMN superoxide production in response to phorbol myristate acetate (PMA) (14% and 23%, respectively; P less than .05). Whereas 1-14 was found to increase PMA-induced cell degranulation significantly (175%), Ab 1-15 did not alter degranulation response to PMA. Immunoprecipitation showed that Ab 1-14 and Ab 1-15 recognized respective surface antigens of 94,000 mol wt and 130,000 to 180,000 mol wt. Our findings suggest that the surface molecules identified by these two monoclonal antibodies play a significant role in neutrophil activation by both FMLP and PMA.     

Effect of chloramphenicol on in vitro function of lymphocytes.

The effect of chloramphenicol on the in vitro function of human peripheral blood lymphocytes was studied in assays of lymphocyte transformation and lymphokine production. When lymphocytes were stimulated by phytohemagglutinin, concanavalin A, or pokeweed mitogen in the presence of various concentrations of chloramphenicol, only minimal effects on blastogenesis were noted. However, suppression by chloramphenicol of blastogenesis induced by candida antigen or streptokinase-streptodornase was greater in magnitude and was dose-dependent; blastogenesis was suppressed to 25%--30% or normal levels by concentrations of chloramphenicol of 25--50 microgram/ml. Chloramphenicol had little effect on the production of the lymphokine leukocyte migration inhibition factor by lymphocytes stimulated either by candida antigen or by concanavalin A, whereas puromycin at a concentration of 5 microgram/ml significantly suppressed this response. Thus chloramphenicol appears to suppress antigen-induced lymphocyte blastogenesis significantly but not lymphokine production by stimulated lymphocytes.     

In vitro and ex vivo effects of indobufen on human platelet aggregation, the release reaction and thromboxane B2 production

We have done a comprehensive study in normal volunteers of the in vitro and ex vivo effects of the antiplatelet agent indobufen on platelet aggregation, the release reaction and thromboxane B2 (TxB2) production as induced by different concentrations of aggregating agents. At low concentrations (10 microM), indobufen completely inhibited secondary platelet aggregation, the release reaction and TxB2 production stimulated by ADP, epinephrine and low concentrations of platelet-activating factor (PAF acether). Higher concentrations of indobufen (100 microM) completely inhibited TxB2 production, platelet aggregation and ATP release induced by arachidonic acid (1 mM) or collagen (2 micrograms/ml). The inhibitory effect was partially overcome by higher concentrations of arachidonic acid (2 mM). Data obtained ex vivo 2 h after the oral administration of 200 mg indobufen to 8 normal volunteers were in keeping with those of the in vitro study. We conclude that indobufen inhibits platelet aggregation and the release reaction by inhibiting the platelet arachidonate pathway.     

K562 cells produce an anti-inflammatory factor that inhibits neutrophil functions in vivo.

We have previously reported that K562, a chronic myelogenous leukemia cell line, releases a low molecular weight factor (6 to 8 Kd) that inhibits human polymorphonuclear neutrophil (PMN) adherence and adherence-related functions tested in vitro. We now report that this factor, which we have named K562 inhibitory factor (K562-IF), has potent anti-inflammatory activity in mice, associated with an inhibition of PMN functions. Its in vitro actions were less marked with mouse PMN than with human PMN. They included (1) an inhibition of both nonstimulated locomotion and locomotion induced by FMLP or serum; (2) an inhibition of the chemiluminescence induced by opsonized zymosan, but not that induced by phorbol myristate acetate or FMLP; (3) an inhibition of the degranulation stimulated by opsonized zymosan, as reflected by lactoferrin and lysozyme release; and (4) a decrease in arachidonic acid release and leukotriene B4 production by A23187-stimulated PMN. The in vivo actions of K562-IF after intraperitoneal injection included (1) an inhibition of subcutaneous PMN accumulation at the site of injection of opsonized zymosan (PMN accumulated neither outside the vessels nor intravascularly, as shown by means of histochemistry); (2) an inhibition of neutrophil accumulation in the peritoneum of mice having received sodium caseinate or opsonized zymosan intraperitoneally; and (3) lysozyme concentration in neutrophils having reached the peritoneum after opsonized zymosan treatment equal to that in blood, suggesting diminished release. PMN influx and degranulation in the peritoneum were reduced by 50% after 3 hours of treatment with 1 microgram of K562-IF (equivalent to the effect of 120 micrograms of prednisolone). Taken together, these results show that K562-IF is a potent anti-inflammatory agent that acts by inhibiting PMN functions.     

Mepacrine but not methylprednisolone decreases acute edematous lung injury after injection of phorbol myristate acetate in rabbits

A good model of the adult respiratory distress syndrome (ARDS) is intravenously injected phorbol myristate acetate (PMA), which causes pulmonary sequestration of neutrophils and a neutrophil-dependent acute edematous lung injury in rabbits. In the present study, pretreatment of rabbits with the antimalarial agent, mepacrine, prevented lung edema after injection of PMA without altering initial accumulations of neutrophils in the lung. Mepacrine also decreased oxygen radical production and degranulation by neutrophils stimulated by PMA in vitro. In contrast, pretreatment with methylprednisolone did not decrease edematous lung injury in rabbits given PMA nor did it inhibit O2 radical production or degranulation by neutrophils treated with PMA in vitro. Our results suggest that agents that modify neutrophil function may be useful in decreasing lung injury after PMA treatment and, perhaps, in treating patients with ARDS.     

Impaired granulocyte formylpeptide-induced superoxide generation in chronically ill, malnourished, elderly patients

OBJECTIVE: Protein-energy malnutrition (PEM), often occurring in patients with chronic disease, is associated with a decreased capacity to combat infections. In this study we assessed polymorphonuclear neutrophil (PMN) superoxide anion (*O2-) formation in elderly PEM patients with various chronic diseases. DESIGN AND SUBJECTS: Nineteen patients (75+/-1 years), with body mass index 17.1+/-0.4, and 19 age-matched healthy controls were included. Fourteen patients and 14 controls were re-examined in a 3 month follow-up. SETTING: A service of internal medicine at a university-affiliated hospital. INTERVENTIONS: Eight patients were prescribed a dietary liquid supplementation during the observation period. MAIN OUTCOME MEASURES: Superoxide production in PMN induced by fMLP (a receptor ligand) and phorbol myristate acetate (PMA), which acts directly on protein kinase C. RESULTS: fMLP-induced superoxide generation in the malnourished patients was 55+/-5% of that of the controls. However, the patients retained their capacity, 108+/-6% of control PMN generation, to respond to PMA. In those who received formula supplementation, fMLP-generated *O2- production levels were 48+/-8 and 73+/-13% (P = 0.12) of those of controls at the start and after 3 months, respectively. Corresponding figures in those who were not prescribed supplementation were 57+/-8 and 64+/-4% (P = 0.55). CONCLUSION: Possibly contributing to reduced host defence, receptor ligand-induced PMN generation of *O2 is significantly lower in chronically ill, elderly patients with PEM than in age-matched healthy controls.     

Enhanced superoxide generation and the decreased superoxide scavenging activity of peripheral blood leukocytes in Beh?et's disease--effects of colchicine.

Enhanced superoxide radical (O2-) generation by polymorphonuclear cells (PMN) has been suggested to mediate tissue damage associated with Beh?et's disease (BD). Because superoxide dismutase (SOD) provides protection from O2- we investigated whether the superoxide scavenging activity (SSA) of PMN, mononuclear cells (MNC) or plasma has a correlation to the amount of O2- generated by PMN in BD. We also studied the effects of colchicine, a potent inhibitor of phagocytosis, on the SSA of PMN. In BD, O2- release by both non-stimulated PMN and those stimulated with opsonized zymosan (OZ), or 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA), was enhanced. The SSA of PMN, but not MNC and plasma, was significantly lower in BD patients as compared with healthy controls. The SSA of PMN showed a strong negative correlation with their O2- release. The SSA of PMN was lower in the BD patients with elevated erythrocyte sedimentation rates and C-reactive protein levels than in those with normal test results. In addition, we found that colchicine treatment increased the SSA of PMN toward normal levels in BD patients. In vitro studies demonstrated that the SSA in PMN obtained from healthy adults was decreased after stimulation with either OZ or PMA. Colchicine could prevent the decrease in the SSA when PMN were stimulated with OZ, but not when stimulated with PMA. Our results suggest that the enhanced O2- release by PMN in vivo may be responsible for the decreased SSA of PMN in BD and the PMN might be able to release more O2- in tissues. Colchicine may increase the SSA of PMN toward normal levels by blocking phagocytosis.