Generation of chemiluminescence by a particulate fraction isolated from human neutrophils. Analysis of molecular events.

A particulate fraction isolated from human neutrophils by homogenization, then centrifugation at 27,000 g, was demonstrated to generate chemiluminescence. This luminescence required the addition of reduced pyridine nucleotide and was very low in fractions from resting normal cells. Stimulation of neutrophils with opsonized zymosan, phorbol myristate acetate, or ionophore A23187 resulted in marked enhancement of the chemiluminescence measured in subsequently isolated particulate fractions. Stimulation did not boost the luminescence produced by fractions from cells of patients with chronic granulomatous disease. The chemiluminescence of particulate fractions from stimulated neutrophils was linear with increasing protein concentration, had a pH optimum of 7.0, and was higher with NADPH as substrate than with NADH. These results confirm previous studies suggesting that the enzyme system responsible for the respiratory burst in neutrophils is present in this fraction. The particulate fraction was used to examine the nature and origin of neutrophil luminescence by investigating the effect on this phenomenon of certain chemical and enzymatic scavengers of oxygen metabolites. Results suggest that the energy responsible for the luminescence of particulate fractions and, presumably, the intact cell, is derived from more than one oxygen species and that luminescence is a product of the interaction of these species and excitable substrates within the cell.

Further characterization of NADPH oxidase activity of human polymorphonuclear leukocytes.

Mn2+ was shown to catalyze a nonenzymatic oxidation of NADPH in the presence of superoxide anion by means of an isotopic assay for measurement of the oxidation of NADPH to NADP+. Human polymorphonuclear leukocyte granule NADPH oxidase activity was evaluated in the absence of Mn2+ and was found to be higher in granules from phagocytizing cells than in granules from resting cells. The drug phorbol myristate acetate, which affects the oxidative metabolism of the neutrophil like phagocytosis, was found to activate granule NADPH oxidase activity. Superoxide dismutase was shown to inhibit NADPH oxidase activity both in the presence and absence of added Mn2+. The NADPH oxidase reaction in the absence of Mn2+ was optimal at pH 5.5, and was more linear with increasing time and protein concentration than in the presence of Mn2+. No activity was measurable in granules isolated from resting cells until the level of NADPH added was above 0.25 mM. Activity was present in granules isolated from cells challenged with opsonized zymosan, even at 0.05 mM NADPH, and was higher than the activity found in granule fractions from resting cells at all levels of NADPH tested. The addition of as little as 0.1 muM NADH to the reaction mixture was found to inhibit granular NADPH oxidase activity, indicating a possible regulatory role for NADH. These results suggest that NADPH oxidase may be the enzyme that initiates the metabolic events accompanying phagocytosis.

Comparison of NADH and NADPH oxidase activities in granules isolated from human polymorphonuclear leukocytes with a fluorometric assay.

A fluormetric method for the determination of pyridine nucleotides has been adapted for use in studying the reduced pyridine nucleotide oxidases in human polymorphonuclear leukocytes. In the presence of strong base the oxidized forms of the pyridine nucleotides form a highly fluorescent product. The small amounts of NAD(P) formed by the oxidase reactions can be determined with great sensitivity. This method has been compared to the radioisotopic assay for NADPH oxidation. Both methods gave essentially the same results in terms of nanomoles NADP produced by control, resting, and phagocytizing samples. Both NADPH and NADH oxidase activities were insensitive to cyanide. NADPH oxidation had a pH optimum of 5.5, while that for NADH appeared to be 6.0. Granules isolated from phagocytizing cells routinely showed more activity toward both substrates (two to threefold) than granules from resting cells. Both activities were located primarily in a granule fraction prepared by differential centrifugation. Oxidation of NADPH was routinely four to five times that of NADH at all except very high substrate levels. Measurable NADH oxidation was rarely seen below 0.80 mM NADH, while NADPH oxidation was easily measurable at 0.20 mM. One patient with chronic granulomatous disease was studied. At low substrate levels, there was no activity toward either substrate in granules isolated from either resting or phagocytizing cells of this patient, while granules isolated from normal control cells showed substantial activity at these substrate levels. Purification of the activities had been initiated with linear sucrose gradients. Both activities co-sediment to a very dense region of the gradient, a region different from that in which membrane or azurophil granules usually equilibrate. The peak gradient fractions show a 10-30-fold increase in specific activity over comparable granule fractions. These data suggest that the oxidase activities are associated with one enzyme that has different affinities for the two substrates ans support the contention that the oxidation of NADPH is responsible for the metabolic burst accompanying phagocytosis in human PMNL.

Superoxide dismutase activity in leukocytes.

Superoxide dismutase activity has been identified in both human neutrophils and rabbit alveolar macrophages by two distinct assay procedures. The enzyme is insensitive to both cyanide and azide and is present in the cytosol of the cell. The identification of this enzyme in phagocytic cells is compatible with the theory that superoxide anion might be involved in the bactericidal activity of the cell. It is proposed that the enzyme functions to protect the cell against superoxide generated during the phagocytic process.

Enhanced phagocytic capacity. The biologic basis for the elevated histochemical nitroblue tetrazolium reaction.

The biologic basis for the elevated histochemical reduction of nitroblue tetrazolium dye (NBT) in neutrophils from patients with acute bacterial infection or polycythemia vera was studied. A precipitin reaction followed mixing NBT with heparin. NBT was reduced after phagocytosis of this complex (H-NBT) by polymorphonuclear leukocytes (PMNs). Ingestion required divalent cations and was facilitated by the presence of complement. H-NBT incubated with normal but not with C2-deficient human serum converted native C3 to its inactive form. Phagocytic indices were determined in patients and controls by measuring O(2) utilization and hexose monophosphate shunt activity and by visually counting cell-associated latex particles. Significant elevations above controls were observed in phagocytes isolated from all patients with elevated histochemical NBT scores when H-NBT complex, latex, or zymosan was employed as the phagocytic particle. Increased indices were observed in the presence of fresh AB serum, heat-inactivated AB serum, or without serum. Serum from patients with elevated NBT scores did not alter phagocytosis in control phagocytes. With NADH and NADPH as substrates, total NBT diaphorase activity of sonicated leukocytes was normal in all patients. These results suggest that increased phagocytic capacity of PMNs is the primary cause of increased histochemical NBT reduction. The PMNs of patients with acute bacterial infection or polycythemia vera may have alterations in their cell membranes which lead to an enhanced rate of phagocytosis.

An isotopic assay for NADPH oxidase activity and some characteristics of the enzyme from human polymorphonuclear leukocytes.

An isotopic assay for NADPH ixodase that measures the amount of NADP formed by the 6-phosphogluconate dehydrogenase reaction has been developed. Under appropriate conditions, the amount of NADP present is directly proportional to the amount of 14CO2 released from [1-14C]6-phosphogluconic acid. Because this assay employs radioisotopes, it is far more sensitive than conventional assays for the enzyme. The human granule NADPH oxidase, as measured by this assay, is active in the presence of CN minus, is stimulated by Mn-2+, and has a pth optimum of 5.5. Granules isolated from cells that have been allowed to ingest zymosan consistently exhibited more enzyme activity than did granules isolated from either resting cells or cells challenged with zymosan that was not preopsonized. This effect was observed over a wide range of substrate concentrations and could not be explained by differences in protein concentrations between the various samples. If whole homogenates are used in place of isolated granules, the enzyme activity can be observed only with a homogenate of phagocytizing cells and even then only at a high concentration of NADPH. This suggests that an inhibitor of the enzyme might be present within the cell. One patient with chronic granulomatous disease was studied. There was no difference in tnadph oxidase activity of the patients' cells when granules from resting and phagocytizing cells were compared. In contrast, the enzyme activity in granules from two control patients doubled upon phagocytosis. These results are consistent with a role for NADPH oxidase in the initiation of the respiratory burst accompanying phagocytosis by human neutrophils.

Comparison of human eosinophils from normals and patients with eosinophilia.

Previous studies of the biochemistry and physiology of eosinophils have relied upon cells obtained from patients with eosinophilia (EE). It is unknown whether such cells might have been activated or partially exhausted by the pathological state causing eosinophilia. We examined cell surface charge, membrane transport of deoxyglucose, activation of lyso-somal acid phosphatase, and oxidative metabolism to provide a profile to compare EE with purified normal eosinophils (NE) and normal neutrophils. Eosinophils or neutrophils were obtained in >95% purity from normal individuals and patients with eosinophilia of diverse etiologies. Cell surface charge was determined by electrophoretic mobility in micromoles per second per volt per centimeter. Normal eosinophils demonstrated a surface charge of 2.46+/-0.03. Stimulation of the cells by zymosan-activated serum (ZAS) reduced the surface charge to 1.82+/-0.02. In contrast, the charge of "resting" EE was already reduced (1.89+/-0.05) and was not altered by ZAS. Resting and stimulated neutrophils had a charge of 1.98+/-0.01 and 1.69+/-0.02, respectively. Uptake of [(3)H]2-deoxyglucose has been shown to reflect carrier-facilitated hexose transport in granulocytes. Deoxyglucose uptake by resting NE and NE stimulated by ZAS was 2.40+/-0.40 and 5.44+/-0.39 (cpm x 10(-3)/2 x 10(5) eosinophils), respectively. Resting and stimulated EE demonstrated deoxyglucose uptake of 7.55+/-0.58 and 15.3+/-0.6, respectively.Lysosomal acid phosphatase was determined by an electron microscopic cytochemical technique. In normal eosinophils and neutrophils, lysosomal acid phosphatase in mature cells is held in a latent form. Normal eosinophils demonstrated weakly positive acid phosphatase activity in 7.8+/-1.2% of the specific granules. Normal eosinophils, stimulated by opsonized staphylococci or the calcium ionophore A23187, develop rapid activation of acid phosphatase in approximately 80% of the granules throughout the cells. Resting EE were usually already activated and demonstrated acid phosphatase in 48.6+/-8.6% of the granules (range, 2-95% granules positive; significant activation was observed in preparations in EE from 11 of 15 patients). Oxidative metabolism was monitored by measurement of the hexose monophosphate shunt (HMPS) (metabolism of 1-[(14)C]glucose to (14)CO(2)). Previous studies demonstrated that resting EE have an HMPS activity which is nearly that of stimulated neutrophils, yet EE remain capable of further 7-10-fold increase when stimulated by opsonized zymosan. In contrast, the HMPS of NE (resting and stimulated) was not significantly different from that of neutrophils. Thus eosinophils obtained from patients with eosinophilia appear significantly activated when compared with normal eosinophils by the criteria of surface charge, activation of lysosomal acid phosphatase, membrane hexose transport, and hexose monophosphate shunt activities.

Eosinopenia of acute infection: Production of eosinopenia by chemotactic factors of acute inflammation.

One distinctive aspect of the response to acute inflammation involves a rapid and persistent decrease in the numbers of circulating eosinophils, yet the mechanisms of this eosinopenia are undefined. One possibility is that the abrupt eosinopenia may be the result of release of small amounts of the chemotactic factors of acute inflammation into the circulation. These studies were designed to examine the numbers of circulating eosinophils after an intravenous injection of zymosan-activated serum, partially purified C5a or the synthetic peptide, N-formyl-methionyl-leucyl-phenylalanine. Each of these factors caused a virtual disappearance of circulating eosinophils within 1 min, a transient return of eosinophils to approximately 50% of control levels after 10-90 min, and a subsequent decrease which persisted for 5 h. In contrast, the numbers of circulating heterophils, although dropping transiently, rapidly returned and rose to elevated levels for 6 h after injection. The response was not caused by adrenal mediation as it occurred normally in adrenalectomized rabbits. Two chemotaxins of allergic inflammation, histamine and the tetrapeptide valine-glycine-serine-glutamic acid, did not cause significant eosinopenia. Circulating granulocytes of patients undergoing hemodialysis, which has been reported to activate complement, demonstrated similar eosinopenic and neutropenic-neutrophilic responses. Thus, in rabbits and in man, intravascular activation or injection of chemotactic factors (C5a or N-formyl-methionyl-leucyl-phenylalanine) causes a brief, nonspecific granulocytopenia followed by a prolonged eosinopenic-neutrophilic response analogous to that seen during acute infection.

Complete deficiency of leukocyte glucose-6-phosphate dehydrogenase with defective bactericidal activity.

A 52 yr old Caucasian female (F. E.) had hemolytic anemia, a leukemoid reaction, and fatal sepsis due to Escherichia coli. Her leukocytes ingested bacteria normally but did not kill catalase positive Staphylococcus aureus, Escherichia coli, and Serratia marcescens. An H(2)O(2)-producing bacterium, Streptococcus faecalis, was killed normally. Granule myeloperoxidase, acid and alkaline phosphatase, and beta glucuronidase activities were normal, and these enzymes shifted normally to the phagocyte vacuole (light and electron microscopy). Intravacuolar reduction of nitroblue tetrazolium did not occur. Moreover, only minimal quantities of H(2)O(2) were generated, and the hexose monophosphate shunt (HMPS) was not stimulated during phagocytosis. These observations suggested the diagnosis of chronic granulomatous disease. However, in contrast to control and chronic granulomatous disease leukocytes, glucose-6-phosphate dehydrogenase activity was completely absent in F. E. leukocytes whereas NADH oxidase and NADPH oxidase activities were both normal. Unlike chronic granulomatous disease, methylene blue did not stimulate the hexose monophosphate shunt in F. E. cells. Thus, F. E. and chronic granulomatous disease leukocytes appear to share certain metabolic and bactericidal defects, but the metabolic basis of the abnormality differs. Chronic granulomatous disease cells lack oxidase activity which produces H(2)O(2); F. E. cells had normal levels of oxidase activity but failed to produce NADPH due to complete glucose-6-phosphate dehydrogenase deficiency. These data indicate that a complete absence of leukocyte glucose-6-phosphate dehydrogenase with defective hexose monophosphate shunt activity is associated with low H(2)O(2) production and inadequate bactericidal activity, and further suggest an important role for NADPH in the production of H(2)O(2) in human granulocytes.

Enzymes of phospholipid synthesis in Bacillus Calmette-Guerin induced rabbit alveolar macrophage. Characterization and localization of cytidine diphosphocholine phosphotransferase and monoacylphospholipid acyltransferase.

The rabbit alveolar macrophage is capable of renewing its plasma membrane by at least two metabolic pathways. It contains (1) a monoacylphospholipid acyltransferase, which catalyzes the synthesis of diacylphospholipids by recycling monoacylphospholipids produced by the action of phospholipases and (2) a cytidine diphosphocholine phosphotransferase (CDPcholine phosphotransferase), which catalyzes the last step in the synthesis de novo of diacylglycerophosphocholine. These activities have been characterized in the cell homogenate with respect to time, protein, pH optimum (for CDPcholine phosphotransferase), substrate specificity (for monoacylphospholipid acyltransferase) and cation requirement ( for CDPcholine phosphotransferase). Monoacylphospholipid acyltransferase activity is localized solely in the endoplasmic reticulum. On the other hand, the CDPcholine phosphotransferase activity can be measured in the endoplasmic reticulum and in the plasma membrane, characterized by both differential and gradient sedimentation techniques. In addition to the normal route of phospholipid synthesis in the endoplasmic reticulum, the rabbit alveolar macrophage may thus possess the capacity for in situ synthesis of phospholipids of plasma membrane as a mechanism for membrane renewal following phagocytosis.

Membrane lipid metabolism of Bacillus Calmette-Guerin-induced rabbit alveolar macrophages.

We examined the uptake of radiolabeled lysophospholipids and oleic acid by Bacillus Calmette-Guerin-induced rabbit alveolar macrophages either in the presence or absence of challenge particles. There was no difference in the uptake and metabolism of lysophospholipids by control or challenged cells for incubation periods up to 5 h. When incubated with [3H]oleic acid, challenged cells consistently exhibited a slightly greater uptake of radioactivity. Extraction of the whole cells revealed that the greater amount of radioactivity found in the challenged cells primarily was in triacylglycerol. There was no marked difference in the amount of radioactivity associated with the phospholipids in the whole cell extracts from control and challenged cells. When the macrophages were pre-labeled for 15 min with [3H]oleic acid and then reincubated in fresh medium in the presence or absence of autoclaved Escherichia coli B, more radioactivity was retained by the challenged cells, again in the form of triacylglycerol. Only in isolated plasma membrane fractions did we observe a difference in the amount of radioactivity associated with phospholipids from control and challenged cells. Plasma membranes isolated from Bacillus Calmette-Guerin-induced rabbit alveolar macrophages that had been incubated for 6 h with [3]oleic acid in the presence of E. coli B contained significantly higher level of radioactivity in all lipids than plasma membranes from control cells. Since the greatest and the most consistent difference between control and challenged cells is associated with the triacylglycerol molecule, it is postulated that this molecule may serve as a precursor in the synthesis of alveolar macrophage phospholipids, both by the reacylation pathway and the de novo pathway. It is possible that the high level of radiolabeled phospholipid found in the plasma membrane arose via the de novo pathway following the cleavage of an acyl group as we have found cytidine diphosphocholine phosphotransferase in the plasma membrane fraction (Wang, P., DeChatelet, L.R., and Waite, M. (1977) Biochim. Biophys. Acta 450, 311--321).

Mechanism of arachidonic acid release in human polymorphonuclear leukocytes.

Previous studies have demonstrated that [3H]arachidonic acid is released from prelabeled human neutrophil phospholipids when the cells are stimulated by calcium ionophore A23187 or by opsonized zymosan. Neither lysophospholipid generated by phospholipase A2 activity, diacylglycerol nor monoacylglycerol produced via phospholipase C/diacylglycerol lipase action have been identified following neutrophil challenge. The inability to detect any intermediates during the release of arachidonate is due to either rapid reacylation of lysophospholipid or conversion of diacylglycerol (monoacylglycerol) to cellular acylglycerols. The addition of exogenous [14C]fatty acid at the time of challenge was employed to determine the involvement of either phospholipase A2 or phospholipase C activities. Neutrophil stimulation with calcium ionophore A23187 resulted in an incorporation of exogenous [14C]arachidonate into phosphatidylinositol and phosphatidylcholine, those phospholipids which specifically release arachidonate. When the saturated fatty acid, [14C]stearate, replaced [14C]arachidonate, very little [14C]fatty acid was incorporated into any of the phospholipid species. Lipid phosphorus measurements revealed no significant mass change in any phospholipid class following ionophore challenge. Production of [14C]phosphatidic acid was not detected, as would be expected if diacylglycerol kinase and de novo phospholipid metabolism were significantly involved.

Role of Ca2+ and Mg2+ in neutrophil hexose transport.

The influence of extracellular Ca2+ and Mg2+ on the transport of 2-deoxy-[3H]glucose into human polymorphonuclear neutrophils was studied. Omission of these cations from the cell suspensions had little effect on resting hexose uptake. Furthermore, the addition of the bivalent cation chelator, EDTA, depressed uptake only slightly. Similarly, neither cation was essential for the enhanced 2-deoxy-D-[3H]glucose uptake stimulated by two chemotactic factors (C5a and N-formylmethionylleucylphenylalanine) and arachidonic acid: enhanced uptake was only partially depressed by the omission of Ca2+ and Mg2+ from the suspensions and was still prominent in the presence of EDTA. Two other neutrophil stimulants, the ionophores, A23187 and ionomycin, also enhanced hexose uptake but their actions were heavily dependent upon extracellular bivalent cations and were totally abrogated by EDTA. In all instances, extracellular Ca2+, but not Mg2+, supported optimal enhanced hexose transport induced by stimuli. Activation of 2-deoxy-D-[3H]glucose uptake by each of the five stimuli was totally blocked by cytochalasin B (a blocker of carrier-mediated hexose transport) and D-glucose but not by L-glucose. The data indicate, therefore, that a variety of neutrophil stimulants activate carrier-mediated hexose transport. Although this transport can be triggered by the movement of extracellular Ca2+ into the cell (as exemplified by the action of the two ionophores), such Ca2+ movement is not required for the actions of chemotactic factors or arachidonic acid. Other mechanisms, such as a rearrangement of intracellular Ca2+, may be involved in mediating the activation of hexose transport induced by the latter stimuli.

Identification and quantitation of electron-transport components in human polymorphonuclear neutrophils.

Using dithionite difference spectra we have detected cytochrome b in highly purified human neutrophils at a concentration of 0.08 nmol/mg protein. The presence of quinone was identified in lipid extracts at a concentration of approx. 0.06 nmol/mg protein. It was identified as ubiquinone-10 by mass spectrographic analysis. Simultaneous measurements of cytochrome oxidase indicated that these compounds could not be attributed to mitochondrial contamination. These results are compatible with the hypothesis that initiation of the respiratory burst in human neutrophils involves a multicomponent electron-transport system.

Oxidation of glucosamine by human polymorphonuclear leukocytes.

When exposed to a phagocytic stimulus (opsonized zymosan), human polymorphonuclear leukocytes (PMNs) produced 14CO2 from [1-14C]glucosamine at a rate 10-25% of that produced from glucose under the same conditions. The production of CO2 from glucosamine by intact PMNs was inhibited by glucose and dependent upon activation of the hexosemonophosphate shunt (HMPS). However, the metabolic pathways for the oxidation of glucose and glucosamine by PMNs are not identical. This is suggested by the fact that glucose-6-phosphate dehydrogenase, the initiating enzyme for the HMPS, did not utilize glucosamine-6-phosphate as a substrate. In addition, glucosamine was not oxidized by sonically disrupted PMNs whereas oxidation of glucose by the same preparation was increased sevenfold over intact cells. Taken together, the data suggest that PMNs oxidize glucosamine by converting it to a compound compatible with the enzymes of the HMPS. This conversion requires intact PMNs and/or an as yet unidentified cofactor.

Pyridine nucleotide-dependent generation of hydrogen peroxide by a particulate fraction from human neutrophils: effect of substrate concentration.

NAD(P)H oxidase activity was determined in particulate fractions from human neutrophils by measuring the production of hydrogen peroxide. Activity was measured over a wide range of substrate concentrations from 0.0 to 4.0 mM. The activity with NADPH was consistently greater than with NADH. Activity towards both substrates was higher in a particulate fraction derived from cells which had phagocytized opsonized zymosan than in a corresponding fraction from resting cells. This increased activity was apparently due to a decreased Km of the enzyme, although no evidence of allosteric kinetics was obtained. The activity was markedly reduced in the presence of superoxide dismutase, indicating the involvement of a superoxide-mediated chain reaction. Particular fractions derived from cells of a patient with chronic granulomatous disease exhibited decreased activity towards both substrates and an apparent defect in the activation of the enzyme by phagocytosis.

Oxidation of 2-deoxyglucose by human polymorphonuclear leukocytes.

Human polymorphonuclear leukocytes (PMNL) can metabolize [1-14C]2-deoxyglucose to 14CO2 when stimulated by either phorbol myristate acetate (PMA) or opsonized zymosan. Oxidation of 2-deoxyglucose is about 10-120% as efficient as that of glucose in normal PMNL; it does not occur in defective cells obtained from patients with chronic granulomatous disease. The increased oxidation of [1-14C]2-deoxyglucose induced by PMA is not sufficient to explain the inhibition of transport induced by that compound; conversely increased transport of 2-deoxyglucose induced by zymosan-activated serum does not result in a significant increase in oxidation of the hexose. Oxidation of [1-14C]2-deoxyglucose appears to be mediated by the hexose monophosphate shunt as indicated by the following (1) oxidation of [1-14C]2-deoxyglucose in intermediate in activity between that of [1-14C]glucose and [6-14C]glucose; (2) the reaction is insensitive to cyanide or azide; and (3) shunt enzymes measured in a cell-free extract from human PMNL can react with 2-deoxyglucose compounds with approximately 10% the efficiency shown towards the corresponding glucose derivatives.

Does tuftsin alter phagocytosis by human polymorphonuclear neutrophils?

The physiological significance of the putative phagocytosis-promoting peptide, tuftsin, was investigated by measurement of chemiluminescence generated during phagocytosis and by assay of the uptake of radiolabeled bacteria. We found no differences in either assay when we compared serum from splenectomized patients (which purportedly lacks tuftsin) with normal serum. Further, there was no difference when serum from splenectomized patients was employed in the presence of absence of exogenous tuftsin. Similar results were obtained under a variety of conditions, utilizing three different challenge particles with varying particle-cell ratios and serum from 20 different splenectomized patients. These results do not agree with the hypothesis that tuftsin plays a major role in promoting phagocytosis.

Co-localization of superoxide generation and NADP formation in plasma membrane fractions from human neutrophils.

In order to resolve discrepancies in the literature concerning the subcellular localization of NADPH oxidase, we disrupted human neutrophils by nitrogen cavitation and fractionated the subcellular organelles on a discontinuous sucrose density gradient. The lightest fraction was 20- to 40-fold enriched for plasma membranes as determined by the marker enzymes alkaline phosphatase and phosphodiesterase I as well as by the ratio of lipid phosphorus to protein. There was a significant decrease in the specific activities of the granule markers myeloperoxidase, lysozyme, and beta-glucuronidase. An intermediate fraction was enriched in membrane markers but not to the extent the lightest fraction was enriched. This fraction contained more granular contamination, as shown by the marker enzymes. In contrast, the densest bands of the gradient were enriched for granule markers with little contamination by plasma membrane. Superoxide generation and NADP formation were primarily associated with the two membrane-enriched fractions from polymorphonuclear leukocytes stimulated with phorbol myristate acetate. The NADP formation associated with a dense granule fraction observed previously in our laboratory was probably due to a cyanide-stimulated oxidation of NADPH by myeloperoxidase.

Neutrophil responses to platelet-activating factor.

1-O-Alkyl-2-O-acetyl-sn-glycerol-3-phosphorylcholine (i.e., platelet-activating factor) was prepared and confirmed to possess potent platelet aggregating activity. It was also potent in aggregating and degranulating rabbit and human neutrophils. When injected into rabbits, the lipid induced profound neutropenia, thrombocytopenia, and anaphylactic symptoms. The lyso derivative of this lipid, 1-O-alkyl-sn-glycerol-3-phosphorylcholine, was inactive or several orders of magnitude weaker in inducing these responses. The acetylated lipid appears to be a potent stimulator of both platelets and neutrophils. Its anaphylactic-like toxicity may be related, at least in part, to its ability to aggregate or otherwise stimulate these cells.