Role of myeloperoxidase in respiratory burst of human polymorphonuclear leukocytes

A comparative study of the respiratory burst [monitored as superoxide (O₂ ^–) production] of normal and myeloperoxidase (MPO) -deficient poiymorphonuclear leukocytes (PMNs) was carried out on 11 MPO-deficient subjects that represent the largest sample of this kind ever studied. The rate of O₂ ^– production by isolated PMNs and whole blood from normal and MPO-deficient subjects was comparable during the initial 30–40 min of incubation with serum-treated zymosan (STZ). Afterwards, the amount of O₂ ^– produced became progressively higher in MPO-deficient cells at least until 120 min incubation with STZ. On the contrary the rate of O₂ ^– production by both cell types in response to 4--phorbol-12-myristate-13-acetate (PMA) was the same. The PMNs of four MPO-deficient subjects were tested for their ingestion ability by counting the number of ingested particles on toluidine blue-stained sections of epoxy-embedded PMN suspensions. Both cell types ingested STZ particles at a comparable rate at early postphagocytic times, whereas on prolonged incubation MPO-deflcient PMNs ingested more STZ particles than normal PMNs. These results suggest that the ingestion capacity of normal cells may undergo a more rapid deterioration than that of MPO-deficient cells during incubation with STZ. Evidence for a higher deterioration of normal PMNs with respect to MPO-deficient PMNs was obtained also from studies on the effect of storage on O₂ ^– generation. After standing at melting ice temperature for 3 h, normal PMNs produced less O₂ ^– than MPO-deficient PMNs in response to PMA, and the difference in O₂ ^– production by the two cell types in response to STZ was evident at earlier postphagocytic periods than with freshly isolated cells. Taken all together these results suggest that normal PMNs and MPO-deficient PMNs do not intrinsically differ in O₂ ^– generating potential and that the difference in the respiratory burst observed during phagocytosis may be accounted for by a more marked deterioration, in normal PMNs, of one or more functions related to the respiratory burst.     

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.     

Human phagocytic cells as oxygen metabolite scavengers

Human neutrophils or monocytes decreased hydrogen peroxide (H₂O₂) concentrationsin vitro. Neutrophils or monocytes decreased H₂O₂ concentrations as well as human erythrocytes. Treatment with aminotriazole or azide decreased both phagocyte and erythrocyte catalase activity and the ability of each cell to decrease H₂O₂ concentrationsin vitro. Prestimulation of phagocytic cells with phorbol myristate acetate (PMA) or opsonized zymosan decreased neither their catalase activity nor their ability to decrease H₂O₂ concentrations. The results suggest that unstimulated or stimulated phagocytic cells can scavenge H₂O₂ and may potentially decrease H₂O₂-mediated tissue injury. The H₂O₂ scavenging potential of phagocytic cells is due at least partially to their catalase activity.This work was supported in part by grants from the National Institutes of Health (P50 HL40784), Johnson and Johnson, Council for Tobacco Research Inc., Procter and Gamble, and Tambrands, Inc.     

Role of myeloperoxidase in the host defense against fungal infection]

Neutrophils are believed to be the first line of defense against invading microorganisms, but in vivo roles of reactive oxygens produced by neutrophils are not well known. Myeloperoxidase (MPO) catalyzes reaction of hydrogen peroxide with chloride ion to produce hypochlorous acid that is used for microbial killing by phagocytic cells. To define the in vivo role of MPO, we generated mice having no peroxidase activity in their neutrophils or monocytes. MPO-deficient (MPO-KO) mice showed severely reduced cytotoxicity to Candida albicans, Aspergillus fumigatus, Cryptococcus neoformans, and other microorganisms, demonstrating that an MPO-dependent oxidative system is important for host defense against fungi. However, the significance of MPO compared to the NADPH-oxidase is still unclear because individuals with MPO deficiency are usually healthy in contrast to patients with chronic granulomatous disease (CGD) who present clinical symptoms early in life. To better understand the contributions of MPO and NADPH-oxidase to antifungal defense mechanisms, we compared the susceptibility of MPO-KO mice and CGD mice to infections by C. albicans. Interestingly, at the highest dose, the mortality of MPO-KO mice was comparable to CGD mice, but was the same as normal mice at the lowest dose. These results suggest that MPO and NADPH-oxidase are equally important for early host defense against a large inocula of Candida. Our present results suggest that MPO-deficient individuals could exhibit similar problems as CGD patients if exposed to a large number of microorganisms.     

Effect of sodium azide on hydrogen peroxide production by zymosan-activated human neutrophils

Stimulated neutrophils (PMNs) produce large quantities of superoxide anion, which is the precursor for hydrogen peroxide (H₂O₂). We developed a new fluorimetric assay to measure the H₂O₂ released by zymosan A-activated PMNs utilizing the oxidation ofp-hydroxyphenylacetic acid by H₂O₂ to its fluorescent dimer in the presence of horseradish peroxidase. Zymosan-activated PMNs isolated from nine healthy volunteers and 20 patients with acute hypoxemic respiratory failure (AHRF) released after 90 min 2.3±0.3 and 2.4±1.3 nmol H₂O₂/10⁶ PMNs, respectively. Inhibition of the heme enzymes by 1.0 mM sodium azide (NaN₃) increased the H₂O₂ production to 21.6±4.4 nmol H₂O₂/10⁶ PMNs in the control group (P<0.001), and to 22.5±14.7 nmol H₂O₂/10⁶ PMNs in patients with AHRF (P<0.001). Incubation temperature, room temperature or 37C, did not change the total amount of H₂O₂ produced after 90 min by zymosan-activated PMN. Addition of NaN₃ improved both the sensitivity and reproducibility of the measurement of H₂O₂ and allowed detection of H₂O₂ released by PMNs with coefficients of variation of less than 5% at PMN concentrations as low as 0.1×10⁶ cells/ml. The amount of H₂O₂ released by activated PMNs did not distinguish healthy controls from patients with AHRF.     

Genetic disorders of leukocyte function: What they tell us about normal antimicrobial mechanisms of human phagocytic cells

Summary Analysis of three inherited defects of granulocyte function (Chediak-Higashi Syndrome, CHS; Chronic Granulomatous Disease, CGD; Myeloperoxidase Deficiency, MPO) has highlighted critical events for the antimicrobial function of these cells and placed others in perspective. Prompt phagosomal fusion may be more important for digestion of organisms rather than killing as indicated by the mild bactericidal defects in the CHS. The formation of O₂- and H₂O₂ during the phagocytic respiratory burst is central for the broad antimicrobial activity of granulocytes. MPO, on the other hand, while perhaps normally participating in granulocyte antimicrobial action, appears to be essential only for the effective killing of eukaryotic organisms such as certain fungal strains. While the non-oxidative killing mechanism of neutrophils have stimulated much recent interest and were the first to be defined no specific inherited defects have been discovered which are clinically important.Genetic disorders of macrophage effector function remain to be clearly defined as do those of eosinophils. The lessons learned from the study of the granulocyte defects discussed have provided both the technology and approach to the analysis of the antimicrobial and cytocidal mechanisms of these important phagocytic cells.Supported in part by USPHS No. 5 ROI AI13251     

Protective and inactivating effects of neutrophil myeloperoxidase on C1q activity

This study investigated the interaction between neutrophil myeloperoxidase (MPO) and the C1q component of the complement system. Using a dot-spot assay, MPO was found to bind to C1q in a dose-dependent manner. The specificity of this reaction was proved by the inhibitory effect of F(ab')2 antibodies to C1q and by the inability of MPO to bind to Clr, Cls and IgG. The interaction between MPO and C1q did not influence the enzymatic activity of the peroxidase but resulted in a more stable C1q as assessed by hemolytic assay for C1q. The protective effect of MPO on C1q did not require the presence of H₂O₂ in the reaction mixture nor was it inhibited by sodium azide, whereas it was abolished by heating the peroxidase. Lactoferrin and lysozyme, unlike MPO, were ineffective in protecting C1q from functional decay. Addition of H₂O₂ and chloride to MPO and C1q led to a complete inactivation of C1q, which could not be induced by H₂O₂ alone. The hypochlorite, which is known to be generated during the reaction of MPO with H₂O₂ and chloride, exhibited a similar inactivating effect on C1q, which was prevented by an external source of methionine.     

Deficiency in C3b receptors on neutrophils of patients with chronic granulomatous disease and hyperimmunoglobulin-E recurrent infection (Job's) syndrome

C3b receptor (CR1) expression by neutrophils (PMNs) and erythro-cytes (Es) from patients with chronic granulomatous disease (CGD) or with hyper-IgE, frequent infection (Job's) syndrome was compared with that of control subjects. The control subjects consisted of one group of patients with infections and a second group of normal, healthy individuals. Three quantitative assays were used: rosette formation with C3b-coated cellular intermediates (EAC43b), binding of radiolabeled monoclonal anti-CRl ([¹²⁵I]anti-CRl) to PMN surfaces, and binding of the antibody to nonidet P-40 (NP-40) extracts of PMNs and Es in an immunoradiometric assay. Rosette formation by the PMNs of five male CGD patients was about 50% of that of paired normal control subjects, whereas the rosette formation of three female CGD patients was similar to that of the control subjects. Surface binding of [¹²⁵I]anti-CRl to PMNs of 10 CGD patients was about half that of the normal subjects (mean percent binding was 2.33% for the CGD patients vs. 3.86% for the normal subjects, giving a difference of -1.53 ± 0.22%,P < 0,001 by the paired-samplet test). The degree of PMN binding was similarly low for both the male and the female CGD patients. Conversely, the binding of anti-CRl to the PMNs of 11 infected control patients appeared to be similar to that of the normal subjects (4.51% for the patient vs. 4.21% for the paired normal subjects). The infected control group originally included four Job's syndrome patients, and when this subgroup was analyzed separately, their PMNs were shown to bind significantly less anti-CRl than did the PMNs of the normal subjects (P < 0.01 by the paired-samplet test). In contrast, the other infected control patients showed higher-than-normal levels of anti-CR 1 binding (P < 0.05). When compared to that of the normal subjects, the total CR1 quantitated in PMN extracts was also lower than normal in CGD patients (P < 0.01 and in the PMN extracts of eight Job's syndrome patients tested (P < 0.01). The PMNs of the other infected control subjects were not significantly different from those of the normal subjects in total CRI expression. Extracts of Es from Job's syndrome patients also had fewer than normal CR1 (P < 0.02). On the other hand, CR1 levels in E extracts from the CGD patients and the other control patients were similar to those in the normal control subjects. Quantitations of C 3, C4, and factor B were normal in CGD. Significant levels of immune complexes were detected in serum samples from several CGD and Job's syndrome patients. However, the level of immune complexes did not correlate significantly with the CR1 deficit in CGD or Job's syndrome patients. Thus, in CGD patients, CRI expression of PMNs is below normal, while that of Es is normal; in Job's syndrome patients, both the PMN and the E CRI expression are below normal. These abnormalities do not appear to result from the frequent infections that occur in these diseases since the other infected control patients exhibit an above-normal amount of surface PMN CR1.     

Scavengers of reactive oxygen intermediates do not mediate the depression of macrophage hydrogen peroxide production caused by erythrocyte phagocytosis

Our previous studies have shown that a phagocytic challenge with IgG-coated erythrocytes (EIgG) depressed macrophage triggered H₂O₂ production in vitro, and in vivo there was a decrease in the survival rate following bacteremia. The phagocytosis of an equal number of IgG-coated erythrocyte ghosts had none of these effects, indicating that the contents of the erythrocytes are important for these effects. The present study evaluated the role of the scavengers of reactive oxygen intermediates within erythrocytes in the depression of H₂O₂ production triggered with phorbol myristate acetate following a phagocytic challenge with EIgG. Elicited rat peritoneal macrophages (PM) were challenged with EIgG prepared from normal E or E with inactivated catalase, depleted glutathione, hemoglobin converted to methemoglobin, or fixed with formaldehyde. The depression of triggered H₂O₂ production was similar when equal numbers of normal EIgG and EIgG with inactivated scavengers were phagocytized. When the phagocytic challenge with normal EIgG was carried out in the presence of cytochalasin B, no depression of triggered H₂O₂ production was observed. Cytochalasin B partially blocked the phagocytosis of EIgG, so that with larger doses of EIgG there was sufficient ingestion of EIgG to depress H₂O₂ production in untreated PM. These results indicate that the scavengers of reactive oxygen intermediates present in erythrocytes are neither required nor sufficient to depress H₂O₂ production by macrophages.     

Effect of deuterium oxide on neutrophil oxidative metabolism,phagocytosis, and lysosomal enzyme release

We have previously shown that deuterium oxide (D₂O) enhances the oxidation of methionine, a myeloperoxidase (MPO)-mediated reaction, by human neutrophils during phagocytosis. However, D₂O has no effect on the oxidation of methionine by the purified MPO-H₂O₂-Cl^– system. To explain this observation, we studied the effect of D₂O on the oxidative metabolism, phagocytosis, and lysosomal enzyme release by human neutrophils. D₂O stimulated the hexose monophosphate shunt (HMS) activity of resting neutrophils in a dose-response fashion. In the presence of latex particles or phorbol myristate acetate (PMA), D₂O brought about an exaggerated stimulation of the HMS activity. This enhancement of the HMS activity by D₂O was markedly reduced when neutrophils form two patients with X-linked chronic granulomatous disease (CGD) were used, either in the presence or absence of latex particles or PMA. Superoxide and H₂O₂ production by neutrophils in the presence of latex particles or PMA were also stimulated by D₂O. In contrast, D₂O inhibited the ingestion of latex particles. D₂O enhanced the extracellular release of MPO, but not lactate dehydrogenase, by neutrophils only in the simultaneous presence of cytochalasin B and latex particles. The enhancement of HMS activity and MPO release by D₂O was partially inhibited by colchicine. Our results suggest that enhancement of neutrophil oxidative metabolism by D₂O may in part explain the stimulation of methionine oxidation by phagocytosing neutrophils.     

Impaired host defence against Mycobacterium avium in mice with chronic granulomatous disease

Patients with chronic granulomatous disease (CGD), an inherited disorder of phagocytic cells, often contract recurrent life‐threatening bacterial and fungal infections. CGD is considered to arise from a functional defect of the O₂‐generating nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in phagocytes. To determine whether or not NADPH oxidase is crucial to the host defence against Mycobacterium avium, we investigated the response against M. avium using CGD model mice (gp91‐phox^‐) of C57BL/6 strain. A tracheal injection of 1 × 10⁷ colony‐forming units (CFU)/head of M. avium strain FN into the CGD mice resulted in a pulmonary infection, while also increasing the mortality rate. In contrast, normal C57BL/6 mice injected with same dose of the organisms did not develop severe pulmonary infection and were able to survive through 2 months of observation. The macrophages obtained from the CGD mice were observed to have a higher burden of the bacterial growth than macrophages from normal C57BL/6 mice. These results suggest that the defect of the NADPH oxidase function impairs the host defence against M. avium infection.     

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.     

Interaction of Aspergillus fumigatus Spores with Human Leukocytes and Serum

Serum was necessary for optimal phagocytosis of Aspergillus fumigatus spores by human leukocytes, and its opsonic capacity was greatly diminished by heat inactivation (56 C, 30 min). A germination assay, described in this report, was developed to study the fate of phagocytized spores. After incubation for 3 hr with normal leukocytes and serum, spores ingested by peripheral blood neutrophils and monocytes remained viable. Since we had previously found that myeloperoxidase (MPO), a lysosomal enzyme of human neutrophils and monocytes, exerted fungicidal activity against Candida albicans when combined with H₂O₂ and chloride or iodide, the effects of these substances on A. fumigatus spores were examined. Spore viability was not impaired by MPO alone, H₂O₂ alone, or KI alone, but high concentrations of KI and H₂O₂ in combination caused marked inhibition of subsequent germination. MPO imparted fungicidal activity to concentrations of KI and H₂O₂ that lacked any effect in its absence. NaCl, in combination with MPO and H₂O₂, was far less effective than the iodide salt against A. fumigatus. The relative ineffectiveness of chloride in this system could underly the apparent inability of human neutrophils to kill ingested A. fumigatus spores, despite their competence to kill C. albicans.     

Hydrogen peroxide enhances phagocytosis of Pseudomonas aeruginosa in hyperoxia

Mechanical ventilation with hyperoxia is a necessary treatment for patients with respiratory distress. However, patients on mechanical ventilation have increased susceptibility to infection. Studies including ours have shown that reactive oxygen species (ROS), generated by exposure to prolonged hyperoxia, can cause a decrease in the phagocytic activity of alveolar macrophages. Hydrogen peroxide (H₂O₂) is a form of ROS generated under hyperoxic conditions. In this study, we examined whether treatment with H₂O₂ directly affects macrophage phagocytic ability in RAW 264.7 cells that were exposed to either 21% O₂ (room air) or 95% O₂ (hyperoxia). Moderate concentrations (ranging from 10 to 250 µM) of H₂O₂ significantly enhanced macrophage phagocytic activity and restored hyperoxia-suppressed phagocytosis through attenuation of hyperoxia-induced disorganization of actin cytoskeleton and actin oxidation. These results indicate that H₂O₂ at low–moderate concentrations can be beneficial to host immune responses by improving macrophage phagocytic activity.     

Clinical manifestation of myeloperoxidase deficiency

Myeloperoxidase (MPO), an iron-containing heme protein localized in the azurophilic granules of neutrophil granulocytes and in the lysosomes of monocytes, is involved in the killing of several micro-organisms and foreign cells, including bacteria, fungi, viruses, red cells, and malignant and nonmalignant nucleated cells. Despite the primary role of the oxygen-dependent MPO system in the destruction of certain phagocytosed microbes, subjects with total or partial MPO deficiency generally do not have an increased frequency of infections, probably because other MPO-independent mechanism(s) for microbicidal activity compensate for the lack of MPO. Infectious diseases, especially with species of Candida, have been observed predominantly in MPO-deficient patients who also have diabetes mellitus, but the frequency of such cases is very low, less than 5% of reported MPO-deficient subjects. Evidence from a number of investigators indicates that individuals with total MPO deficiency show a high incidence of malignant tumors. Since MPO-deficient PMNs exhibit in vitro a depressed lytic action against malignant human cells, it can be speculated that the neutrophil MPO system plays a central role in the tumor surveillance of the host. However, any definitive conclusion on the association between MPO deficiency and the occurrence of cancers needs to be confirmed in further clinical studies. Clinical manifestations of this disorder depend on the nature of the defect; an acquired abnormality associated with other hematological or nonhematological diseases has been occasionally described, but the primary deficiency is the form more commonly reported. Another area of interest pertinent to MPO expression is related to the use of anti-MPO monoclonal antibodies for the lineage assignment of acute leukemic cells, the definition of FAB M0 acute myeloid leukemia, the identification of biphenotypic acute leukemias, and their distinction from acute leukemia with minimal phenotypic deviation. The advantage of MPO monoclonal antibodies over the MPO cytochemical assay relies in the ability of the former method to recognize the enzymatically inactive precursor forms of MPO. Received: 22 January 1998 / Accepted: 8 June 1998     

Lysosomal enzymes and metabolic activity of polymorphonuclear leukocytes from patients with systemic lupus erythematosus and from experimental animals after levamisole treatment

The activity of -d-glucuronidase (BDG) was lowered and the activity of myeloperoxidase (MPO) was elevated in polymorphonuclear leukocytes (PMNs) of SLE patients compared with normal subjects. After levamisole treatment, the activities of BDG, MPO, and lysozyme rose in PMNs of SLE patients. A higher activity of lysozyme was also observed in peritoneal PMNs of rabbits after levamisole administration. During incubation of human phagocytizing and non-phagocytizing PMNs, no effects of levamisole at concentrations of 10^–3 to 10^–5 M were observed on the release of lysosomal enzymes. These concentrations of levamisole also did not influence INT reductase activity and production of superoxide by normal human PMNs in the presence of zymosan particles. These findings suggest that levamisole might have an effect on the actual level of lysosomal enzymes in PMNs rather than on their release.     

Immunomodulatory effects of the methanolic extract from Pouteria campechiana leaves in macrophage functions

The present study aimed to examine the immunomodulatory properties of the methanolic (MeOH) extract from Pouteria. campechiana leaves in peritoneal macrophages of Balb/c mice. Peritoneal macrophages isolated from mice and Vero cells were treated with the MeOH extract from leaves. Cell viability of the macrophages and Vero cells were evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide method. The phagocytic activity, as nitric oxide (NO), hydrogen peroxide (H₂O₂), interleukin 6 (IL-6) and tumour necrosis factor α (TNF-α) production were evaluated on peritoneal macrophages. Results showed that the MeOH extract from leaves was able to stimulate the phagocytic activity and increase NO, H₂O₂ and cytokines production. The viability assays do not show cytotoxic effect on cell viability and cause a significative proliferative effect in the macrophages of a concentration-dependent manner. These results conclude that the MeOH extract from P. campechiana leaves possessed a stronger immunostimulatory effect in a concentration-dependent manner without affect the cell viability.     

Comparison of group I and II soluble phospholipases A2 activities on phagocytic functions of human polymorphonuclear and mononuclear phagocytes

Soluble phospholipase A₂ (PLA₂) purified from rheumatoid synovial fluid (group II) and repurifiedNaja naja venom PLA₂ (group I) were compared for their influence on phagocytic activity of human polymorphonuclear (PMN) and mono-nuclear (MO) phagocytes. Group II PLA₂ reduced chemotaxis, adhesiveness, and intracellular bactericidal activity (ICBA) and induced release of muramidase from PMNs. Group I PLA₂ suppressed chemotaxis, and enhanced ICBA but had no influence on other phagocytic functions. Group II PLA₂ purified from synovial fluid or from placenta caused marked spontaneous superoxide generation followed by inhibition of phagocytosis-induced burst of energy. Group INaja naja and porcine pancreatic PLA₂ had no effect on superoxide generation. Group II but not group I PLA₂ reduced markedly ICBA of monocytes. It may be concluded that human group II soluble PLA₂, in concentrations comparable to those present in inflamed joints or in sera of patients with active arthritis or septic shock, causes spontaneous formation of the oxygen radical superoxide and release of lysosomal enzymes, and suppresses conventional phagocytic activities of PMNs and monocytes. Marked differences between group I and group II PLA₂s may mean that these enzymes exert different influences on cell membrane.     

α-MSH对脂多糖部分生物学活性的影响

目的:探讨α-黑色素细胞刺激素对LPS部分生物学活性的影响。方法:本文应用比色法、倒置生物显微镜及流式细胞仪观察小鼠腹腔巨噬细胞释放H₂O₂、中性粒细胞凋亡及FITC-LPS与单核细胞的结合情况。结果:LPS可刺激小鼠腹腔巨噬细胞释放H2O2,而α-MSH与LPS共同培养,则能明显抑制巨噬细胞释放H₂O₂(P<0.01);α-MSH及LPS本身均不影响中性粒细胞凋亡(P>0.05),但在LPS作用下,α-MSH可显著促进中性粒细胞凋亡(P<0.01);并且,α-MSH可降低FITC-LPS与单核细胞的结合率及单核细胞表面的平均荧光强度(P<0.05,P<0.01)。结论:α-MSH不仅能有效抑制LPS刺激巨噬细胞释放H₂O₂、促进LPS作用下的中性粒细胞发生凋亡;而且可干扰LPS与单核细胞的结合,发挥其有效的免疫调控作用。     

Enhancement of normal neutrophil chemiluminescence by chronic granulomatous disease neutrophils

Neutrophils and other phagocytic cells from patients with chronic granulomatous disease (CGD) lack the ability to generate reactive oxygen intermediates (ROI), although other phagocytic functions appear to be intact. The effects of CGD neutrophils on the ability of normal neutrophils to produce ROI as measured by luminol-enhanced chemiluminescence (CL) were examined. Normal neutrophils (2×10⁵) had a peak CL response to phorbol myristate acetate (PMA; 20 ng/ml) of 6.5±0.9 mV, while the CL response of CGD neutrophils was completely absent. However, the addition of CGD neutrophils (8×10⁵) to normal neutrophils (2×10⁵) markedly increased the peak CL response to PMA to 11.0+-1.1 mV (P < 0.001). The peak response of normal neutrophils (2×10⁵) alone to the peptideN-formyl-methionyl-leucyl-phenylalanine (FMLP; 10^–6 M) was 9.0±1.1mV, and this increased to 22.2±3.5 mV (P < 0.001) when 8×10⁵ CGD neutrophils were added and to 18.9±3.6 mV (P<0.005) when 4×10⁵ CGD neutrophils were added. Thus, CGD neutrophils increase the release of ROI from normal cells, suggesting nonoxidative regulatory factors in ROI production.This work was supported in part by grants GI517-02 and 0008-08 from the Cystic Fibrosis Foundation and HD-09800 and AI-15332 from the National Institutes of Health.