Lawrence R. DeChatelet

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.

A modified procedure for the determination of loukocyte alkaline phosphatase

Abstract Although the determination of serum alkaline phosphatase in a glycine buffer is a standard procedure, this buffer is totally unsuitable for assaying the leukocyte enzyme. The leukocyte enzyme is almost totally inhibited by preincubation in 0.1 m glycine for 5 minutes; hence the reaction is not linear with time when run in this buffer. A satisfactory assay for the leukocyte enzyme employing 2-amino-2-methyl-1-propanol buffer is outlined.

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.

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.

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.

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.

Phagocytosis-induced release of arachidonic acid from human neutrophils

Abstract The phospholipids of human neutrophils were labeled with [3H] arachidonic acid and [14C] palmitic acid. Phagocytosis of opsonized zymosan resulted in rapid release of free arachidonic acid but not of palmitic acid. Arachidonic acid was not released when the cells were exposed to unopsonized zymosan, zymosan-activated serum, or phorbol myristate acetate. These observations suggest that phagocytosis of opsonized zymosan results in the activation of a phospholipase A2.

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.

Deficiency of NADPH oxidase activity in chronic granulomatous disease

NADPH oxidase activity was examined in paired 27,000 x g granule fractions isolated from normal polymorphonuclear leukocytes from patients with chronic granulomatous disease. At 0.17 mM NADPH, the oxidase activity was not measurable in normal resting cells but was activated by phagocytosis. This activation was absent in CGD cells. At higher levels of NADPH, activity was present in cells from patients with CGD, although it was lower than normal, and no difference in activity was found between resting and phagocytizing cells. Granule fractions from phagocytizing normal cells exhibited higher than granule fractions from resting normal cells at all levels of NADPH. These results suggest that NADPH oxidase activity is defective in chronic granulomatous disease, and further that the defect is not the absence of the enzyme but rather a failure to activate it.

Human eosinophilic peroxidase: Biochemical characterization

Abstract In the present communication, peroxidases from the neutrophil and eosinophil have been compared. The biochemical characteristics of the enzymes were strongly dependent on the method of assay employed. Using four different hydrogen donors, neutrophil and eosinophil peroxidases were found to have different comparative activities. With the hydrogen donor o-dianisidine, both enzymes showed similar activity. Using o-tolidine and guaiacol, eosinophil peroxidase was somewhat more active. There was a dramatic sixfold increase of eosinophil peroxidase above the neutrophil enzyme using p-phenylenediamine as the hydrogen donor. Both enzymes were inactivated by excess H2O2. The pH optimum for eosinophil and neutrophil peroxidases was 5.5 and 7.0 using o-dianisidine and p-phenylenediamine, respectively. Cyanide and azide inhibited both enzymes, the degree of inhibition being dependent on the hydrogen donor used.