Neutrophil responses to platelet-activating factor

1-O-Alkyl-2-O-acetyl-sn-glyceryl-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-glyceryl-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.This work was supported by NIH grants AI09169, AI10732, AI14929, HL16769, HL14164, and AMI1799.     

Phagocytosis of Live Versus Heat-Killed Bacteria by Human Polymorphonuclear Leukocytes

Heat-killed Pseudomonas aeruginosa are phagocytized much more slowly by human polymorphonuclear leukocytes than are the live organisms. The post-phagocytic increase in hexose monophosphate shunt activity (HMS) parallels the ingestion of the bacteria. The addition of serum to the live organisms causes a marked increase in both ingestion and cellular HMS activity; serum actually causes an inhibition of both uptake and HMS activity when added to the heat-killed organisms. Differences in postphagocytic HMS activity between live and heat-killed organisms were observed with three different species of bacteria, indicating that the phenomenon is not restricted to P. aeruginosa. These data emphasize that the influence of the particle on the phagocytic process is considerable.     

Oxidative metabolism of the human eosinophil

We have compared the oxidative metabolism of human eosinophils (80%-90% purity) to that of neutrophils. Hexose monophosphate (HMP) shunt activity of eosinophils was higher than that of neutrophils under either resting or phagocytizing conditions. Eosinophil HMP shunt activity also was stimulated by phorbol myristate acetate, a membrane- active agent. Eosinophils showed a marked incorporation of 125I into trichloroacetic acid-insoluble material under resting conditions, which increased markedly during phagocytosis. Eosinophils likewise showed a greater reduction of nitroblue tetrazolium dye during phagocytosis than did neutrophils. Measurement of other parameters of oxidative metabolism indicated that eosinophils generated superoxide anion following phagocytosis and also elicited a burst of chemiluminescence similar to that observed during phagocytosis by neutrophils. Measurement of NADPH oxidase activity demonstrated that this enzyme was 3-6 times more active in fractions isolated from eosinophils than in corresponding fractions isolated from neutrophils; this was observed over a range of substrate concentrations. The eosinophil enzyme sedimented differently than the neutrophil enzyme with differential centrifugation; neither showed sedimentation characteristics of peroxidase. These data indicate that eosinophils possess a similar, although in some ways more potent, oxidative burst than neutrophils and are consistent with a role for NADPH oxidase in the initiation of that burst.     

Role of arachidonic acid in stimulation of hexose transport by human polymorphonuclear leukocytes

Whereas insulin does not stimulate hexose transport in polymorphonuclear leukocytes, we recently reported that C5a causes the leukocytes to take up 2-[³H]deoxyglucose. We now find that fMet-Leu-Phe, in a concentration-related manner with an EC₅₀ (concentration producing 50% of stimulatory activity) of 1.2 nM, causes a 5.5-fold stimulation of deoxyglucose uptake. Moreover, arachidonic acid (5,8,11,14-eicosatetraenoic acid) similarly stimulated deoxyglucose uptake with an EC₅₀ of 0.6 μM. Stimulation by arachidonic acid exhibited structural specificity; five structural analogues of arachidonic acid, including arachidonyl alcohol, 8,11,14-eicosatrienoic acid, 11,14,17-eicosatrienoic acid, 5,8,11,14-eicosatetraynoic acid, and arachidic acid, did not stimulate deoxyglucose uptake. Release and metabolism of arachidonic acid may also be involved in the stimulation of deoxyglucose uptake by fMet-Leu-Phe. Inhibitors of arachidonic acid metabolism (5,8,11,14-eicosatetraynoic acid, nordihydroguaiaretic acid, indomethacin, aspirin, and benzylimidazole) caused parallel changes in the responses to both arachidonic acid and fMet-Leu-Phe. Stimulation of deoxyglucose uptake of polymorphonuclear leukocytes by chemotactic factors or arachidonic acid had the characteristics of carrier-facilitated hexose transport. The response was saturable with increasing concentrations of stimulant or substrate (deoxyglucose). It was stereospecific (inhibited by D-glucose but not by L-glucose) and was inhibited in resting and stimulated cells by 5 μg of cytochalasin B per ml. It was separable from the stimulation of oxidative metabolism; it occurred normally in polymorphonuclear leukocytes from a patient with chronic granulomatous disease (these are incapable of an oxidative metabolic response to membrane stimuli). Thus, stimulation of polymorphonuclear leukocytes is associated with enhanced hexose transport. Moreover, carrier-facilitated hexose transport and arachidonic acid metabolism may be linked, at least in these leukocytes: arachidonic acid mimies the stimulatory effects of chemotactic factors, and blockade of arachidonic acid metabolism inhibits the stimulation of hexose transport by these agents.     

Creatine phosphokinase activity in rabbit alveolar macrophages

An enzyme is present in extracts of rabbit alveolar macrophage which can catalyze the reaction of creatine phosphate with adenosine diphosphate to form adenosine triphosphate and creatine. The enzyme is moderately activated by reduced glutathione, has a pH optimum between pH 6.5 and 7.0, and shows an absolute requirement for Mg(2+). The K(m) for creatine phosphate is approximately 3.6 mm while the K(m) for adenosine diphosphate is about 1.1 mm. The enzyme may play a role in the energy balance of the cell by creating a reserve of energy in the form of creatine phosphate.     

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.     

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.     

The Activated Phagocyte of Polycythemia Vera

Leukocytes isolated from patients withpolycythemia vera (PCV), a panmyelopathy, have increased metabolicactivity during the resting and phagocytizing states. Phagocytes from patients with PCV were studied by counting particle ingestion, measuring hexose monophosphate shunt (HMP)activity by the conversion of glucose-1-¹⁴C to ¹⁴CO₂, and determiningO₂ consumption and nitroblue tetrazolium (NBT) reduction. Phagocytosisof polystyrene particles was increased.Resting glucose-1-¹⁴C activity was 6.9± 2.7 nmoles of glucose oxidized perhour per 5 x 10⁶ phagocytes in normaland 16.4 ± 7.6 nmoles in polycythemiavera phagocytes. Hexose monophosphate shunt following phagocytosis increased to 50.6 ± 10.4 nmoles in normal and 91.7 ± 17.0 in polycythemiavera phagocytes (p < 0.005). Oxygenconsumption was 3.6 ± 0.2 µl/hr/5 x10⁶ phagocytes in resting and 11.4µl/hr/5 x 10⁶ phagocytes in stimulatedcontrols as compared to 4.8 ± 0.2 µlin resting and 18.4 µl in phagocytizingPCV cells (p <0.001). The reductionof NBT by leukocytes was increasedin all resting polycythemia vera phagocytes as compared to control phagocytes. The cells from some patientshad increases in the amount of NBTreduced during phagocytosis. Phagocytes from four severly infected patients had increases in HMP activityin both resting and phagocytizing cellssimilar to those found in noninfectedPCV phagocytes. Although the increased metabolic activity associatedwith phagocytosis can be explainedby increased particle ingestion, the increased activity of resting PCV cellssuggests other metabolic abnormalitiesperhaps related to the age of thephagocyte.

Submitted on August 30, 1971 Revised on April 18, 1972 Accepted on May 6, 1972     

Effects of Promethazine-Hydrochloride on Human Polymorphonuclear Leukocytes

Promethazine hydrochloride at a concentration of 0.033 mg/ml has pronounced effects on leukocyte metabolism and function. The drug inhibits the phagocytosis-induced increases in O(2) consumption and hexose monophosphate shunt activity. Associated with these effects is an inhibition of the iodination of zymosan particles and an inhibition of bacterial killing by the cell. At least two mechanisms appear to be involved. Many of the effects can be explained by an inhibition of phagocytosis, but promethazine also inhibits the decarboxylation of amino acids and iodide fixation in a cell-free system, indicating a specific effect on metabolism. These results may partially account for the action of the drug in ameliorating the effects of erythroblastosis.