Priming of human neutrophil functions by tumor necrosis factor: Enhancement of superoxide anion generation,degranulation, and chemotaxis to chemoattractants C5a and f-Met-Leu-Phe

Recombinant human tumor necrosis factor- (rTNF) stimulated increased generation of Superoxide anion (O ₂ ^– ) by human neutrophils in a concentration-dependent fashion. Preincubation of human neutrophils with rTNF (2.2–2200 units/ml) for 10 min enhanced the subsequent generation of O ₂ ^– in response to C5a and f-MetLeu-Phe(FMLP). Recombinant TNF did not enhance O ₂ ^– generation by neutrophils stimulated with phorbol myristate acetate (PMA). Recombinant TNF alone failed to induce release of myeloperoxidase (MPO) and lysozyme by neutrophils. However, it did enhance the release of MPO and lysozyme by neutrophils stimulated with C5a and FMLP, but not with PMA. Although rTNF alone (0.001–50,000 units/ml) was not chemotactic for neutrophils, preincubation of neutrophils with rTNF (0.001–0.1 units/ml) enhanced the chemotactic activity of suboptimal concentrations of C5a (0.1 nM) and FMLP (5 nM). Neutrophils treated with high concentrations of rTNF (100–10,000 units/ml) showed inhibition of random movement and of chemotaxis induced by C5a or FMLP. We conclude from these studies that rTNF primes neutrophils for enhanced responses to subsequent stimuli and thus may augment the inflammatory response by increased oxidant production and lysosomal enzyme release and promote down-regulation of chemotactic movement.     

Oxidation of amino acids by human neutrophils

We studied the oxidation of alanine and methionine by human neutrophils. Phagocytosis enhanced the decarboxylation of amino acids by human neutrophils. Decarboxylation of amino acids was dependent on the myeloper-oxidase system (MPO-H₂O₂-Cl^–). This was further confirmed using purified canine MPO. Human neutrophils and the MPO system were about 10 times more efficient in decarboxylating alanine than methionine. They also oxidized methionine to methionine sulfoxide. The fraction of methionine decarboxylated by human neutrophils or the MPO system was small compared to the fraction which was oxidized to methionine sulfoxide. Thus methionine was preferentially oxidized to methionine sulfoxide by the MPO system. However, once methionine was oxidized to methionine sulfoxide, it was readily decarboxylated by the MPO system. The results suggest that the thio group of methionine prevents its carboxylic group from being decarboxylated.     

In vivo latex phagocytosis primes the Kupffer cells and hepatic neutrophils to generate superoxide anion.

Activation of liver macrophages during clearance of endotoxins, bacteria, or other particulate materials may be accompanied by the migration of polymorphonuclear neutrophils (PMNs) into the liver and priming of the hepatic phagocytes to release toxic oxygen metabolites. In the present study we investigated the effect of in vivo administration of latex particles on the hepatic sequestration of PMNs and the release of superoxide anion (O2-) by the in situ perfused rat liver and isolated hepatic phagocytes. One hour after an intravenous injection of latex beads, a significant amount of O2- (0.7 nmol/min/g) was produced by the in situ perfused liver. Administration of latex particles into the perfused liver also elicited O2- production. Hepatic phagocytes from latex-treated rats generated large amounts of O2- (2-14 nmol/60 min/10(6) cells) when these cells were stimulated in vitro with opsonized zymosan or phorbol myristate acetate (PMA), whereas phagocytes from saline-treated rats released less than 0.8 nmol O2-. Intravenous infusion of superoxide dismutase or ibuprofen did not prevent the immigration of PMNs to the liver. However, ibuprofen inhibited the production of O2- by the perfused liver. Also, after addition of ibuprofen in vitro to isolated cells, there was more than 50% inhibition of O2- generation by Kupffer cells and hepatic PMNs treated with either zymosan or PMA. These observations suggest that arachidonic acid metabolites play a role in O2- release under these conditions. Thus, activation of the reticuloendothelial system by latex phagocytosis induces the migration of PMNs into the liver and enhances the production of toxic oxygen-derived radicals by these cells and the resident Kupffer cells. The toxic oxygen radicals may also contribute to hepatic injury.     

Inhibition of neutrophil derived lysosomal enzymes and reactive oxygen species by a novel tetrapeptide

OBJECTIVE AND DESIGN: The role of a tetrapeptide derivative PEP 1261 {Boc-Lys(Boc)-Arg-Asp-Ser(tBu)-OtBu}, corresponding to residues 39-42 of human lactoferrin, has been tested in vitro in the modulation of neutrophil function. MATERIAL AND SUBJECTS: The level of non-enzymatic mediators of inflammation such as reactive oxygen species (ROS), enzymatic mediators such as myeloperoxidase (MPO) and lysosomal enzymes have been assessed in the presence or absence of PEP 1261 in phorbol 12-myristate 13 acetate (PMA) stimulated human neutrophils (n = 6) and also in neutrophils isolated from adjuvant induced arthritic rats (AIA) (n = 4). TREATMENT: PEP 1261, at a concentration of 0.14 mM, was added to the neutrophil cultures. STATISTICAL METHOD: The results were analysed by nonparametric statistics using Mann Whitney U test. Results: Addition of PEP 1261 effectively blocked the H2O2 and O2*- release, decreased the levels of MPO levels (p< 0.01) and lysosomal enzymes (p < 0.05) as compared to PMA stimulated human neutrophils. PEP 1261 was also observed to inhibit the levels of H2O2, O2*-, MPO and lysosomal enzymes (p < 0.05) as compared to PMA stimulated control rat neutrophils and neutrophils from arthritic rats. CONCLUSIONS: The results of this study indicate that PEP 1261 could serve as an excellent antiinflammatory agent.     

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.     

Prevention of peroxidase mediated inhibition of neutrophil motility and lymphocyte transformation by levamisole, OMPI, sodium aurothiomalate, indomethacin and tolmetin in vitro

The effects of sodium aurothiomalate, levamisole, its active metabolite OMPI and the anti-inflammatory agents indomethacin and tolmetin on neutrophil motility and post-phagocytic hexose monophosphate shunt activity, superoxide and H2O2 generation and myeloperoxidase (MPO) mediated iodination of Candida albicans were investigated in vitro. All five agents caused stimulation of neutrophil random motility and migration towards the leucoattractants f-met-met-phe and EAS. Only levamisole caused inhibition of H2O2 and superoxide production, which was associated with inhibition of HMS activity and not related to superoxide scavenging activity. All five agents caused inhibition of MPO mediated iodination of C. albicans. The relationship between inhibition of peroxidase mediated iodination and enhanced motility was further investigated using the horseradish peroxidase (HRP) H2O2/iodide system. Incubation of neutrophils with this system caused inhibition of neutrophil motility. However in the presence of the various drugs neutrophils were protected from inhibition of motility by the HRP/H2O2/iodide system. Further experiments showed that lymphocyte transformation to mitogens was also inhibited by the HRP/H2O2/iodide system. Incubation of lymphocytes with the various drugs prior to exposure to HRP/H2O2/iodide protected the lymphocyte mitogenic responsiveness.     

Dissociation of phagocytosis,metabolic stimulation and lysosomal enzyme release in human leukocytes

In this paper studies are reported concerning the relationship between particle binding to the plasma membrane of human polymorphonuclear leukocytes (PMN's), phagocytosis, generation of oxidative metabolites, and the release of lysosomal enzymes by these cells. Superoxide (O ₂ ^– ) generation by, and lysosomal enzyme release from normal PMN's and cytochalasin B-treated cells were measured. We have found that neither phagocytosis nor lysosomal degranulation are prerequisites for enhanced O ₂ ^– generation. Cytochalasin B-treated PMN's, incapable of ingesting particles but still able to bind particles to membrane receptors, generated enhanced amounts of O ₂ ^– when treated with serum-treated zymosan (STZ), a C3b receptor stimulus, or with aggregated IgG (agg IgG), an Fc receptor stimulus. Moreover, the soluble stimulators complement component C5a, phorbol myristate acetate (PMA), and calcium ions in the presence of the ionophore A23187, also increased the O ₂ ^– production of these cells. In all cases a time and dose-dependent stimulation was found of both the O ₂ ^– generation and the lysosomal enzyme release, but there was no correlation between ability of any stimulus to provoke enzyme release and its ability to stimulate O ₂ ^– generation. When PMN's were preincubated with 5×10^–4 M hydrocortisone-Na-succinate, lysosomal enzyme exocytosis with the immune reactants was inhibited 16–35%. Hydrocortisone also inhibited O ₂ ^– generation, except when STZ was used as the stimulus. Thus, in the case of stimulation of functional processes of PMN's via the C3b receptor, hydrocortisone inhibits membrane fusion without interfering with one of the early biochemical events (O ₂ ^– production).Financially supported by a travel grant from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.).     

Phorbol myristate acetate-induced neutrophil autotoxicity

We have previously shown that in the presence of phorbol myristate acetate (PMA), neutrophils kill neoplastic cells as well as themselves. This PMA-induced neutrophil autotoxicity was markedly inhibited by catalase, suggesting that H₂O₂ directly or indirectly played an important role. In this study we compared PMA and H₂O₂ toxicity against human neutrophils. The effect of H₂O₂ was faster and more sensitive to catalase and serum than that of PMA. Sodium azide markedly enhanced the effect of H₂O₂ but not that of PMA. In contrast, methionine and histidine prevented the toxicity of PMA, while they had no effect on H₂O₂ toxicity. The results suggest that PMA-induced neutrophil autotoxicity is not mediated by H₂O₂ alone.     

A New Assay to Monitor the Degranulation Process in Phagocytizing Human Neutrophils

The microbicidal activity of phagocytes depends on intraphagosomal secretion (i.e., the degranulation process), during which the content of the secretory granules is discharged into the phagosome to kill ingested microorganisms. The availability of a reliable assay to quantify the extent of degranulation can be an important tool to gain a deeper insight into the mechanism of bacterial processing in phagocytes and into the transmembrane signaling that leads to granule–phagosome fusion. In an early study, Baehner et al. (25) showed by using the peroxidase chromogenic substrate DAB that the occurence of intraphagosomal MPO release in human neutrophils. Starting from that finding and from that of Herzog and Fahimi (26), who reported the quantitative evaluation of peroxidase-mediated DAB oxidation, we set up a method for measuring MPO intraphagosomal release in human neutrophils. The method is based on the passive engulfment of DAB together with the phagocytosable particle. Inside the vacuole, this substrate is oxidized by MPO released from the azurophilic granules. The colorimetrical evaluation of the amount of DAB oxidized allows for cheap, rapid quantification of MPO intraphagosomal secretion in whole cells. Using this method, we show that the degranulation process, involving azurophilic granules, can be monitored carefully during phagocytosis. It takes place after the ingestion of zymosan particles opsonized with normal human serum, as well as during IgG-mediated phagocytosis and under conditions where 2 integrins are blocked. However our findings also show that the extent of intraphagosomal secretion depends on either the extent of opsonization or the type of receptor engaged during the phagocytic event.     

Engagement of adenosine receptors inhibits hydrogen peroxide (H2O2-) release by activated human neutrophils

Adenosine and its analogs, acting at specific cell surface receptors, inhibit generation of superoxide anion by neutrophils. Since it has been suggested that hydrogen peroxide (H2O2) release may not be contingent upon superoxide anion release, we studied the effects of 2-chloroadenosine, a potent adenosine receptor agonist, on the formation of H2O2 by neutrophils exposed to various stimuli: n-formyl-methionyl-leucyl-phenylalanine (FMLP), concanavalin A, phorbol myristate acetate (PMA), serum-treated zymosan particles (STZ), and immune complexes. 2-Chloroadenosine (0.01-10 microM) inhibited formation of H2O2 by neutrophils exposed to FMLP, concanavalin A, and STZ particles. As we have found with O2- generation, 2-chloroadenosine failed to inhibit H2O2 release by neutrophils stimulated by either phorbol myristate acetate or immune complexes. The data show that whereas adenosine and its analogs inhibit neutrophil release of H2O2 and superoxide anion in response to most ligands, they fail to inhibit activation of neutrophils by immune complexes. Nor do they inhibit neutrophil activation by PMA, an agent which bypasses cell surface receptors by direct activation of protein kinase C. Surprisingly, we found that adenosine deaminase activity was adsorbed onto zymosan particles during opsonization and enhanced release of H2O2 by neutrophils exposed to STZ. These studies with yeast cell walls suggest that if microorganisms adsorb adenosine deaminase from serum, then the intracellular microbicidal activity of neutrophils is enhanced.     

Stimulation of neutrophil oxidative metabolism by indomethacin

Human neutrophils exposed to indomethacin demonstrate an enhanced capacity for superoxide ion (O ₂ ^– ) generation when stimulated with opsonized zymosan. Enhancement is not seen with indomethacin-treated cells exposed to solube oxidative stimuli. To further investigate this phenomenon, O ₂ ^– generation, chemiluminescence, and phagocytosis were assessed in human neutrophils preincubated with indomethacin. Zymosan-stimulated O ₂ ^– release was increased from 150 to 300% of controls in neutrophils exposed to 400 g/ml. indomethacin. Enhancement was not reversed by removal of indomethacin from the medium prior to addition of the stimulus and was dose-dependent at drug concentrations of 5 to 400 /ml. Neutrophils exposed to methacin alone also generated more O ₂ ^– than control cells, although this increment was not sufficient to account for the degree of enhancement seen when indomethacintreated cells were exposed to zymosan. Neutrophil cehmiluminescence induced by zymosan was also increased by exposure to indomethacin, and at a drug concentration of 400 g/ml (1.1 mM), enhancement randed from 253 to 333% of controls. As was observed with O ₂ ^– generation, chemiluminescence of neutrophils was increased in the presence of indomethacin alone, although, to a degree far less than was seen when drug-treated cells were stimulated with zymosan. Phagocytosis of radiolabeledS. aureus by neutrophils incubated with indomethacin was increased 13±5% over controls (P<0.01,n=5), but was unaltered by incubation of cells with the buffer used to solubilize the drug. The modest degree of enhancement of phagocytosis suggests that increased particle uptake is not the sole mechanism of oxidative enhancement. The data are in keeping with the hypothesis that indomethacin has a direct effect on the neutrophil plasma membrane and/or the O ₂ ^– -forming oxidase.     

Myeloperoxidase associated with neutrophil extracellular traps is active and mediates bacterial killing in the presence of hydrogen peroxide

A variety of inflammatory stimuli induces NETs. These structures consist of a network of chromatin strands associated with predominately granule proteins, including MPO. NETs exhibit antimicrobial activity, which is proposed to augment the more-established mechanism of phagosomal killing. They may also be detrimental to the host in situations such as chronic inflammation or severe sepsis. The objective of this study was to establish whether MPO associated with NETs is active and able to kill bacteria. Neutrophils were stimulated with PMA to release NETs. Peroxidase activity measurements were performed and showed that enzymatically active MPO was released from the neutrophils, 2-4 h after stimulation, concomitant with NET formation. Approximately 30% of the total cellular MPO was released, with the majority bound to the NETs. The bound enzyme retained its activity. Staphylococcus aureus were not killed when added to preformed NETs under our assay conditions. However, addition of H(2)O(2) to the bacteria in the presence of NETs resulted in MPO-dependent killing, which was observed with NETs in situ and with NETs when they were removed from the neutrophils by limited DNase digestion. Our results show that the enzymatic activity of MPO on NETs could contribute to antimicrobial activity or tissue injury when NETs are released from neutrophils at sites of infection or inflammation.     

Influence of neutrophil cationic proteins on generation of superoxide by human polymorphonuclear cells during phagocytosis

The cationic proteins from neutrophyl lysosomes have been shown to modulate phagocytic activity of granulocytes. The present study reports the effects of the cationic protein fractions on the generation of O ₂ ^– by human PMNs during phagocytosis. Human PMNs were reacted win different phagocytic stimuli in the presence and absence of lysosomal cationic proteins and the amount of O ₂ ^– generated was determined by superoxide dismutase inhibitable reduction of cytochromec. Total cationic protein extract from neutrophil lysosomes enhanced O ₂ ^– generated by PMNs during the phagocytosis of IgG-coated latex beads and opsonized zymosan particles. The analysis of the fractions of cationic proteins obtained from a Sephadex G-75 column showed that the O ₂ ^– generation-enhancing activity was associated with the proteins eluted in fractions III and IV. A protein fraction mainly eluted in void volume inhibited the cytochromec reduction by O ₂ ^– formed during phagocytosis. This was due to the presence of superoxide dismutase-like activity since O ₂ ^– generated by the xanthine-xanthine oxidase system was also inhibited by this fraction. The cationic protein fractions III and IV from the Sephadex G-75 column were further subfractionated. Although the O ₂ ^– -enhancing activity was eluted in the same fractions as chymotrypsin activity, there was no quantitative correlation between the amount of O ₂ ^– generation and chymotrypsin activity. Moreover, commercial chymotrypsin did not enhance O ₂ ^– generation. Electrophoretic analysis of the isolated protein fractions suggests that O ₂ ^– generation enhancing protein (SGEP) is different from lysozyme or chymotrypsin and probably represents previously undescribed protein.     

Functional comparison of avian heterophils with human and canine neutrophils

Phagocytosis, intracellular calcium flux, oxidant production and bactericidal activity of chicken heterophils and human and canine neutrophils were compared to assess their functional capabilities with respect to resistance of bacterial infection. Five strains of Staphylococcus were compared for bactericidal susceptiblity; two pathogenic strains isolated from chickens with tenosynovitis, a non-pathogenic strain from the tibiotarsal joint of a normal chicken, a pathogenic strain from the femoral joint of a human and a decapsulated derivative of the human strain. As assayed by flow cytometry, latex spheres were phagocytosed by significantly fewer chicken heterophils than by human or canine neutrophils. Phagocytes from all three species responded to stimulation by autologus serum-opsonised zymosan (aOZ) with an intracellular calcium flux, whereas only human neutrophils responded to N-formyl-methionyl-leucyl-phenylalanine (FMLP). None responded to phorbol myristate acetate (PMA). Neutrophils stimulated with aOZ and PMA produced significantly more H₂O₂ than did heterophils. Human neutrophils produced significantly greater H₂O₂ than did either canine neutrophils or chicken heterophils in response to FMLP. Greater bactericidal activity was observed against the decapsulated human strain and the non-pathogenic avian strain; this increased killing was found to be significant with the canine and avian cells. Avian heterophils were less phagocytic and produced less oxidant in response to zymosan than canine and human neutrophils. In addition, heterophils did not respond to PMA or FMLP.     

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.     

Myeloperoxidase-Mediated Oxidation of Methionine and Amino Acid Decarboxylation

The myeloperoxidase (MPO)-mediated decarboxylation of amino acids and the MPO-mediated oxidation of methionine, two potential bactericidal mechanisms, were compared. In the presence of the MPO system (MPO, 50 mU/ml; H(2)O(2), 0.1 mM; Cl(-), 75 mM), 50% of alanine (0.1 mM) was decarboxylated, whereas only 5% of methionine (0.1 mM) was decarboxylated. In contrast, under similar conditions, 80% of methionine was oxidized to methionine sulfoxide. Once methionine was oxidized to methionine sulfoxide, it was decarboxylated (75%) by the MPO system. Methionine at 0.1 mM completely inhibited the decarboxylation of alanine, whereas alanine at a concentration 200 times that of methionine had no effect on the MPO-mediated oxidation of methionine. Sodium azide, an MPO inhibitor, inhibited the decarboxylation of alanine and the oxidation of methionine to the same extent. Tryptophan markedly inhibited the oxidation of methionine, whereas histidine stimulated it. Alanine, glycine, and taurine had no effect. In contrast, all of these amino acids and taurine markedly inhibited the MPO-mediated decarboxylation of alanine. NaN(3), tryptophan, and methionine, which inhibited the MPO-mediated oxidation of methionine, also inhibited the killing of Staphylococcus aureus or Klebsiella pneumoniae by the MPO system; whereas histidine, alanine, and glycine, which did not inhibit the oxidation of methionine, had less or no effect on the killing of these two bacteria by the MPO system. Results suggest that methionine is preferentially oxidized to methionine sulfoxide by the MPO system. Once methionine is oxidized to methionine sulfoxide, it is then readily decarboxylated by the MPO system. The agent responsible for the oxidation of methionine may play an important role in the MPO-mediated killing of bacteria.     

Relationship between phagocytosis and immunoglobulin A release from human colostral macrophages.

Macrophages and neutrophils that contain mainly secretory immunoglobulin A (IgA) comprise the majority of cells in human colostrum. These cell populations were separated and analyzed for their ability to release total IgA and secretory IgA when stimulated to phagocytose. Colostral macrophages phagocytosed opsonized bacteria and nonopsonized latex particles; at the same time, IgA was released. Neutrophils poorly phagocytosed opsonized bacteria but actively phagocytosed latex particles. In contrast to the macrophages, the neutrophils did not release IgA, even after active phagocytosis of latex. Consequently, colostral macrophages are the main source of IgA released from colostral leukocytes when these cells are exposed to organisms or particles that are phagocytosed. A function for colostral neutrophils which sequester IgA is proposed.     

Stimulatory effect of latex and zymosan particles on cyclic adenosine 3',5'-monophosphate content in human granulocytes

Human polymorphonuclear leukocytes respond to latex beads and opsonized zymosan particles with increased cyclic AMP formation. The enhancement of cyclic AMP content in cells is related to particle concentration in the incubation medium and to the time of exposure. The temporary stimulation declines after 15 min of phagocytosis, that is in good relation to the uptake of latex particles which reaches saturation point 10 min after the addition of the beads. The cyclic AMP content in granulocytes decreases after between 15 and 30 min of incubation with latex particles. The decrease is not caused by the loss of the granulocyte viability for: (a) the incorporation of labeled methyldeoxythymidine and uridine into macromolecules continues, (b) the sensitivity of phagocytosing granulocytes to prostaglandin E₁ stimulation of cyclic AMP production persists, (c) less than 10% of cells absorbs trypan blue dye after phagocytosis of latex beads. The prostaglandin synthesis inhibitors, aspirin and indomethacin, have no effect on cyclic AMP content in phagocytosing granulocytes.     

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and opsonization synergistically enhance leukotriene B4 (LTB4) synthesis induced by phagocytosis in human neutrophils

Normal human blood neutrophils were studied for their capacity to synthesize leukotriene B₄ (LTB₄) and its -oxidized metabolites after phagocytosis of zymosan. Phagocytosis of serum-opsonized particles led to a higher release of LTs than did unopsonized zymosan. The most stricking effect of phagocytosis was observed when neutrophils were primed with granulocyte-macrophage colony-stimulating factor (GM-CSF): opsonization and GM-CSF synergistically increased LTB₄ synthesis by neutrophils.     

Phagocytosis by lipopolysaccharide-primed human neutrophils is associated with increased extracellular release of reactive oxygen metabolites

The effect of priming human neutrophils with lipopolysaccharide was investigated regarding the respiratory burst activity generated during phagocytosis of IgG-or C3b-opsonized yeast particles. LPS pretreatment significantly enhanced the respiratory burst activity, measured as luminol-amplified chemiluminescence, of both types of opsonized particles. In control cells most of the activity was produced intracellularly, probably in the phagosomes. In the primed cells, however, extracellular release of reactive oxygen metabolites was significantly increased during Fc-and CR3-mediated phagocytosis (P < 0.01 andP < 0.002, respectively). The release was most pronounced when using C3b-opsonized particles. Potent oxygen metabolites acting together with lysozomal enzymes are of importance in inflammatory-induced tissue damage. An increased extracellular release of reactive oxygen species by phagocytizing primed neutrophils can therefore lead to greater damage to the surrounding tissues.