Fraction Of Neutrophils Research Articles

The neutrophil glycoprotein Mo1 is an integral membrane protein of plasma membranes and specific granules.

The glycoprotein Mo1 has previously been demonstrated to be on the cell surface and in the specific granule fraction of neutrophils and to be translocated to the cell surface during degranulation. It is not known, however, whether Mo1 is an integral membrane protein or a soluble, intragranular constituent loosely associated with the specific granule membrane. Purified neutrophils were disrupted by nitrogen cavitation and separated on Percoll density gradients into four fractions enriched for azurophilic granules, specific granules, plasma membrane, and cytosol, respectively. The glycoproteins in these fractions were labeled with 3H-borohydride reduction, extracted with Triton X-114, and immunoprecipitated with 60.3, an anti-Mo1 monoclonal antibody Mo1 was detected only in the specific granule and plasma membrane fractions and partitioned exclusively into the detergent-rich fraction consistent with Mo1 being an integral membrane protein. In addition, treatment of specific granule membranes with a high salt, high urea buffer to remove absorbed or peripheral proteins failed to dissociate Mo1. These data support the hypothesis that Mo1 is an integral membrane protein of plasma and specific granule membranes in human neutrophils.

A rapid method for the preparation of the neutrophil fraction of granulocytes from human blood by centrifugation on isotonic Nycodenz gradients.

A rapid method for the preparation of neutrophils of the granulocyte fraction of human blood is described. A leucocyte-rich fraction is loaded onto preformed isotonic Nycodenz gradients, formed by mixing a stock isotonic Nycodenz solution with a NaCl diluent solution. After low speed centrifugation a neutrophil fraction of high viability, yield and purity can be isolated from the gradients.

Phorbol myristate acetate and the calcium ionophore A23187 synergistically induce release of LTB4 by human neutrophils: involvement of protein kinase C activation in regulation of the 5-lipoxygenase pathway.

Phorbol myristate acetate (PMA), a tumor-promoting phorbol ester, and the calcium ionophore A23187 synergistically induced the noncytotoxic release of leukotriene B4 (LTB4) and other 5-lipoxygenase products of arachidonic acid metabolism from human neutrophils. Whereas neutrophils incubated with either A23187 (0.4 microM) or PMA (1.6 microM) alone failed to release any 5-lipoxygenase arachidonate products, neutrophils incubated with both stimuli together for 5 min at 37 degrees C released LTB4 as well as 20-COOH-LTB4, 20-OH-LTB4, 5-(S),12-(R)-6-trans-LTB4, 5-(S),12-(S)-6-trans-LTB4, and 5-hydroxyeicosatetraenoic acid, as determined by high pressure liquid chromatography. This synergistic response exhibited concentration dependence on both PMA and A23187. PMA induced 5-lipoxygenase product release at a concentration causing a half-maximal effect of approximately 5 nM in the presence of A23187 (0.4 microM). Competition binding experiments showed that PMA inhibited the specific binding of [3H]phorbol dibutyrate ([3H]PDBu) to intact neutrophils with a 50% inhibitory concentration (IC50) of approximately 8 nM. 1-oleoyl-2-acetyl-glycerol (OAG) also acted synergistically with A23187 to induce the release of 5-lipoxygenase products. 4 alpha-phorbol didecanoate (PDD), an inactive phorbol ester, did not affect the amount of lipoxygenase products released in response to A23187 or compete for specific [3H]PDBu binding. PMA and A23187 acted synergistically to increase arachidonate release from neutrophils prelabeled with [3H]arachidonic acid but did not affect the release of the cyclooxygenase product prostaglandin E2. Both PMA and OAG, but not PDD, induced the redistribution of protein kinase C activity from the cytosol to the membrane fraction of neutrophils, a characteristic of protein kinase C activation. Thus, activation of protein kinase C may play a physiologic role in releasing free arachidonate substrate from membrane phospholipids and/or in modulating 5-lipoxygenase activity in stimulated human neutrophils.

Fucosyltransferase expression in human platelets and leucocytes

The activities of α-2-l-fucosyltransferase and α-3-l-fucosyltransferase were measured in human platelets and leucocytes from normal donors, α-2-l-Fucosyltransferase was found in platelets but not in leucocytes. In contrast α-3-l-fucosyltransferase was not detected in platelets but was present in leucocytes where it was demonstrated in the neutrophil, monocyte and lymphocyte fractions.

A comparative study on the effects of inhibitors of the lipoxygenase pathway on neutrophil function: Inhibitory effects on neutrophil function may not be attributed to inhibition of the lipoxygenase pathway

The effects of five inhibitors of the lipoxygenase pathway were evaluated on oxygen radical production, degranulation, chemotaxis, leukotriene B 4 (LTB 4) production by neutrophils. The lipoxygenase inhibitors tested were nordihydroguaiaretic acid (NDGA), esculetin, eicosatetraynoic acid (ETYA), 2-(12-hydroxydodeca-5,10-diynyl)-3,5,6-trimethyl-1,4-benzoquinone (AA-861), and 6,9-deepoxy-6,9-(phenylimino)-Δ 6.8-prostaglandin I 1 (U-60,257). Neutrophils were activated by n-formyl-methionyl-leucyl-phenylalanine (fMLP), phorbol myristate acetate (PMA), A23187, or platelet activating factor (PAF). The effects of these inhibitors on NADPH oxidase activity and phospholipase A 2 activity of isolated particulate fraction of neutrophils were also evaluated. ETYA inhibited neutrophil function induced by all the stimulators except PMA. AA-681 was unique in that it did not inhibit PAF-induced neutrophil activation. U-60,257 had virtually no effect on oxygen radical production and degranulation, but chemotaxis was moderately suppressed. NDGA effectively inhibited neutrophil function, except for chemotaxis. Esculetin inhibited only oxygen radical production, but this was due to inhibition on NADPH oxidase activity of neutrophil membrane. The inhibitory effect on neutrophil function and that of LTB 4 production were not closely correlated. It is suggested that lipoxygenase inhibitors may modify neutrophil function by the mechanism not involving the lipoxygenase pathway. It is also suggested that LTB 4 may not be a mediator in neutrophil oxygen radical production and degranulation induced by the stimulators used in the present study.

Photolabeling of a O 2⨪ generating protein in bovine polymorphonuclear neutrophils by an arylazido NADP + analog

A plasma membrane fraction of bovine polymorphonuclear neutrophils enriched in NADPH-dependent, O2-. generating oxidase activity, and a number of fractions solubilized in detergent, recovered during the course of the purification of this oxidase have been tested for their ability to react with radiolabeled N-4-azido-2-nitrophenyl aminobutyryl NADP+ (arylazido NADP+ or NAP4-NADP+). In the dark, NAP4-NADP+ and its reduced form NAP4-NADPH, were found to inhibit competitively the NADPH-dependent O2-. generating oxidase activity of the plasma membrane fraction of bovine neutrophils activated by phorbol myristate acetate. The nitrene derivative formed upon photoirradiation of NAP4-NADP(H) bound covalently to different proteins of the plasma membrane. Photolabeling of these proteins was prevented by preincubation with an excess of NADPH. Photolabeling of a protein of 65,000 Mr was decreased by omission of phorbol myristate acetate as activating agent of the respiratory burst in neutrophils or by addition of micromolar amounts of Cibacron Blue and mersalyl which are known to inhibit the production of O2-. by the plasma membrane fraction. During the course of the purification procedure, the 65000 Mr protein emerged as the preferentially photolabeled protein. These data, in agreement with previous findings concerning the purification of an NADPH-dependent, O2-. generating oxidase protein of Mr 65000 from bovine neutrophils (Doussière, J. and Vignais, P.V. (1985) Biochemistry 24, 7231-7239), strongly suggest that a single protein of Mr 65000, located in the plasma membrane fraction of bovine neutrophils, is able to act both as an NADPH deshydrogenase and as an oxygen reductase to generate O2-.

The effect of parainfluenza virus type 3 and pasteurella haemolytica on oxygen-dependent and oxygen-independent bactericidal mechanisms of ovine pulmonary phagocytic cells

Groups of caesarian-derived, colostrum-deprived lambs were inoculated by the intra-tracheal route with Pasteurella haemolytica, either alone or 4 or 6 days after the inoculation of parainfluenza virus type 3 (PI 3). Other groups were inoculated with PI 3 followed by veal infusion broth, or with unifected cell culture fluid followed by veal infusion broth (controls). All lambs were killed 24 h after the second inoculation. Pulmonary phagocytic cells were recovered by lavage and separated into alveolar macrophage (AM) and neutrophil fractions by density gradient centrifugation. Bacterial proliferation was detected in the lungs of all five lambs inoculated with P. haemolytica 6 days after PI 3 but in only one of five inoculated with P. haemolytica 4 days after PI 3 and one of five inoculated with P. haemolytica alone. The number of phagocytic cells recovered from the lungs was highest in animals inoculated with P. h haemolytica 6 days after PI 3 and, overall, a greater number of both AM and neutrophils was recovered from the lungs of animals where bacterial proliferation occurred (>10 5.0 P. haemolytica 100 g −1 lung) than from those that controlled the bacterial infection. Oxygen-dependent bactericidal activity of AM and neutrophils was measured by chemiluminescence. Infection with PI 3 and P. haemolytica increased the chemiluminescence responses. The highest responses were recorded from lambs inoculated with P. haemolytica 6 days after PI 3, the group where pulmonary clearance was poorest. Overall, responses were higher in lambs in which bacterial proliferation occured than in those that controlled the infection. On the other hand, oxygen-independent bactericidal activity, measured by the direct effects of neutrophil lysates on Escherichia coli, was lowest in lambs inoculated with P. haemolytica 6 days after PI 3 and was lower in lambs where bacterial proliferation occurred.

Cytolytic effects of neutrophils: role for a membrane-bound neutral proteinase.

A neutral serine proteinase, purified 250-fold from the plasma membrane fraction of human neutrophils, differs in its catalytic and molecular properties from the well-known neutral proteinases present in azurophil (primary) granules. Stimulation of neutrophils with low concentrations of phorbol 12-myristate 13-acetate (PMA) results in the release into the medium of the membrane-bound proteinase and the concomitant production of oxygen radicals. These concentrations of PMA also induce full cytolytic activity measured with 51Cr-labeled ox erythrocytes. A role for the neutral serine proteinase in the cytolytic activity of PMA-stimulated neutrophils is supported by the following observations: (i) the lytic activity of the stimulated neutrophils is correlated with the quantity of neutral proteinase present in the membranes; (ii) the extracellular medium from PMA-stimulated neutrophils causes the cytolysis of 51Cr-labeled erythrocytes that have been exposed to nonlytic concentrations of H2O2; (iii) cytolysis of H2O2-treated erythrocytes is also observed with the crude proteinase solubilized from neutrophil membranes or with the purified proteinase from the same source; and (iv) in each case the cytolytic activity is proportional to the proteinase activity present and is prevented by the addition of serine proteinase inhibitors. We conclude that cytolysis of target cells by PMA-activated neutrophils can result from the cooperative effects of oxygen radicals and the membrane-bound neutral serine proteinase. The participation of enzymes from specific (secondary) granules is excluded because, with the low concentrations of PMA employed, very little release of secondary granule constituents is observed.

The effect of parainfluenza virus type 3 on the phagocytic cell response of the ovine lung to Pasteurella haemolytica

Groups of Caesarian-derived, colostrum-deprived lambs were inoculated by the intratracheal route with Pasteurella haemolytica 4 or 6 days after the inoculation of parainfluenza virus type 3 (PI 3). Some were killed immediately (0 h) and others 24 h later. Control groups were inoculated with PI 3 alone, P. haemolytica alone or media alone. Pulmonary phagocytic cells, P. haemolytica and PI 3 were recovered by pulmonary lavage. The phagocytes were separated into alveolar macrophage (AM) and neutrophil fractions by density gradient centrifugation and examined biochemically and microbiologically. Twenty-four hours after the inoculation of P. haemolytica bacterial proliferation to greater than 0 h levels had occurred in four of six animals inoculated with P. haemolytica alone, two of eight inoculated with P. haemolytica 4 days after PI 3 and all of eight inoculated with P. haemolytica 6 days after PI 3. Mean bacterial numbers in animals inoculated with P. haemolytica 6 days after PI 3 and killed at 24 h (10 9.1±1.9) were significantly higher than they were in the other two groups killed at this time (PI 3 4 days, P. haemolytica 24 h, mean = 10 5.3±1.7; P. haemolytica alone 24 h, mean = 10 4.5±2.9). Pneumonic lesions were also more severe in the first group. This defect in pulmonary clearance and increase in the severity of pneumonia in animals inoculated with P. haemolytica 6 days after PI 3 coincided with a 1000-fold decrease in virus titres in the lung between Day 6 and Day 7 after virus inoculation and the first detectable evidence of the host's immune response. The virus infection resulted in a significant increase in the number of AM that could be recovered from the lung and an increase in the number of AM with cytoplasmic vacuolation. However, there was no difference in the total number of AM or the number of vacuolated AM between animals that controlled the P. haemolytica infection and those in which proliferation of P. haemolytica occurred. The inoculation of P. haemolytica resulted in a 100-fold increase in the number of neutrophils in the lavage fluid, but there were no differences between virus-infected and uninfected animals, nor was there a difference between animals that controlled the P. haemolytica infection and those that did not. Levels of acid phosphatase, β-glucuronidase and total protein in lavage fluid were increased in animals inoculated with P. haemolytica 6 days after PI 3 and killed at 0 and 24 h; levels of lactate dehydrogenase were also increased in these animals. Therefore, the increased amounts of lysosomal enzymes probably reflected increased cell damage rather than changes in phagocytic activity. When lysates of purified neutrophils and AM were examined there were no differences between groups in the levels of acid phosphatase, β-glucuronidase and total protein. However, the bactericidal activity of neutrophil lysates was significantly reduced in animals inoculated with P. haemolytica 6 days after PI 3, the group where the major defect in clearance was observed.

Identification of precursor of phagocytosis-stimulating factor in guinea pig polymorphonuclear neutrophils

A precursor of phagocytosis-stimulating factor (PSF) was identified by immunoblot assay with purified anti-PSF antibodies. The purified anti-PSF antibodies recognized not only the PSF with a molecular weight of 16,000 (16K) but also the 36K protein with a pI of 6.5 in the granule fraction of polymorphonuclear neutrophils, indicating that this 36K protein has an antigenic determinant common to PSF. In addition, the appearance of the PSF during phagocytosis by polymorphonuclear neutrophils was closely correlated to the decrease of the 36K protein. These results suggest that the 36K protein is the precursor of PSF which is converted to biologically active PSF in the granules during phagocytosis.

Spectrally abnormal cytochrome-b in a male patient with chronic granulomatous disease

Cytochrome-b and flavin adenine dinucleotide (FAD) were measured in neutrophils from a male patient and his family with the X-linked form of Chronic Granulomatous Disease (CGD). The membrane fraction of the patient's neutrophils contained 93pmol/mg protein of FAD (control 102±20, mean±sd), and 32pmol/mg protein of cytochrome-b (control 88±20, mean±sd). In the spectrophotometric study, the maximal absorbance peak of cytochrome-b was at 421nm, as that from control was at 427nm. Cytochrome-b in neutrophils from his mother and a sister, who were thought to be carriers, had their maximal absorbance peak at 425nm. These findings suggest that the spectral abnormality of cytochrome-b contribute to the pathogenesis of CGD in this case.

Detection of lactoferrin and myeloperoxidase release from single neutrophils by a protein A plaque assay.

A haemolytic plaque assay was adopted to detect release of lactoferrin and myeloperoxidase (MPO) from single neutrophils. Target erythrocytes coated with protein A were bound as a monolayer by poly-L-lysine to the surface of a plastic dish. Secreted lactoferrin and MPO induced plaque formation dependent on the reaction with complement and specific antiserum producing lysis of the protein-A-coated sheep red blood cells. Lactoferrin was found to be released spontaneously from a fraction of neutrophils while MPO was released only after phagocytosis, reflecting different mechanisms for degranulation of MPO-containing azurophil and lactoferrin-containing specific granules.

Genetic control of hematopoietic kinetics revealed by analyses of allophenic mice and stem cell suicide

The pluripotential hematopoietic stem cell is influenced by at least one gene that differs between DBA/2 and C3H/He, and C57BL/6 inbred mouse strains. This gene(s) manifests itself by its effect on susceptibility to killing of spleen colony forming cells (CFU-S) caused by hydroxyurea (HU). In strains DBA/2 and C3H/He 20% of the CFU-S population is normally in S phase whereas practically none from strain C57BL/6 are synthesizing DNA. On the other hand, in C57BL/6↔DBA/2 allophenic mice we observed that the proportion of DBA/2 erythrocytes was higher than the proportion of DBA/2 lymphocytes; the fraction of platelets and neutrophils with the DBA/2 genotype fell between the values for erythrocytes and lymphocytes. Control experiments using mice congenic at the Fv-2 locus confirm that in both situations we are examining effects of a gene(s) other than Fv-2. For the effect on the S phase fraction of CFU-S, we refer to the gene(s) as Stk (stem cell kinetics). We suggest that the observed skewing in composition among the various mature blood cell types in C57BL/6↔DBA/2 allophenic mice is caused by allelic variants of the Stk gene. Such variation would favor the formation of DBA/2 erythrocytes, platelets, and neutrophils over those of the C57BL/6 genotype.

Enzymatic deacylation of the lipid A moiety of Salmonella typhimurium lipopolysaccharides by human neutrophils.

Lipid A, the toxic moiety of Gram-negative bacterial lipopolysaccharides (endotoxins), is a glucosamine disaccharide to which fatty acid and phosphate residues are covalently attached. Recent studies of Salmonella lipid A indicate that 3-hydroxytetradecanoic acid (3-OH-14:0) residues are directly linked to the glucosamine backbone and the nonhydroxylated fatty acids (principally dodecanoic and tetradecanoic acids) are esterified to the hydroxyl groups of some of the 3-OH-14:0 molecules. We report here that the granule fraction of human neutrophils contains one or more enzymes that partially deacylate Salmonella typhimurium lipid A by removing the nonhydroxylated fatty acids, leaving almost all of the 3-OH-14:0 residues linked to glucosamine. The available evidence suggests that similar reactions also occur in living neutrophils that ingest lipopolysaccharides by antibody-dependent phagocytosis.

Properties of NADH-cytochrome-b5 reductase from human neutrophils

An NADH-ferricyanide reductase activity of ca. 170 nmole ferricyanide reduced/min/10(7) cells is present in the membrane fraction of human neutrophils. This membrane-bound activity constitutes ca. 85% of the total NADH-ferricyanide reductase activity that is present in these cells. The enzyme(s) readily utilize(s) purified cytochrome-b5 from beef liver as an electron acceptor. No other physiologic electron acceptors tested (e.g., ubiquinone-30, menadione) were active. The specificities of electron donors (e.g., NADH congruent to deamino-NADH much greater than NADPH) and acceptors (e.g., Fe(CN)6-3 greater than 2,6-dichlorophenol-indophenol much greater than O2) for the enzyme(s) in unfractionated membranes, along with action of inhibitors (e.g., ADP, p-chloromercuribenzoate) and the pH optimum, indicate that virtually all of the membrane-bound ferricyanide reductase activity in these cells is NADH-cytochrome-b5 reductase. This reductase, however, is only slightly solubilized (ca. 10%) by a phosphate buffer extraction procedure that is effective with the liver enzyme.

Properties of NADH-Cytochrome-b5 Reductase From Human Neutrophils

Abstract An NADH-ferricyanide reductase activity of ca. 170 nmole ferricyanide reduced/min/10(7) cells is present in the membrane fraction of human neutrophils. This membrane-bound activity constitutes ca. 85% of the total NADH-ferricyanide reductase activity that is present in these cells. The enzyme(s) readily utilize(s) purified cytochrome-b5 from beef liver as an electron acceptor. No other physiologic electron acceptors tested (e.g., ubiquinone-30, menadione) were active. The specificities of electron donors (e.g., NADH congruent to deamino-NADH much greater than NADPH) and acceptors (e.g., Fe(CN)6–3 greater than 2,6-dichlorophenol-indophenol much greater than O2) for the enzyme(s) in unfractionated membranes, along with action of inhibitors (e.g., ADP, p-chloromercuribenzoate) and the pH optimum, indicate that virtually all of the membrane-bound ferricyanide reductase activity in these cells is NADH-cytochrome-b5 reductase. This reductase, however, is only slightly solubilized (ca. 10%) by a phosphate buffer extraction procedure that is effective with the liver enzyme.

Oxidation-reduction properties of the cytochrome b found in the plasma-membrane fraction of human neutrophils. A possible oxidase in the respiratory burst.

The oxidation-reduction midpoint potential of the cytochrome b found in the plasma membrane of human neutrophils has been determined at pH 7.0 (Em,7.0) from measurements of absorption spectra at fixed potentials. In both unstimulated and phorbol myristate acetate-stimulated cells Em,7.0 was -245 mV. Changes in pH affected the Em of the cytochrome b, with a slope of approx. 25 mV/pH unit change. The Em,7.0 of the haem group(s) of the membrane-bound myeloperoxidase of human neutrophils was found to be +34 mV. The plasma membranes contained no detectable ubiquinone, and no iron-sulphur compounds were detected by e.p.r. spectroscopy at 5-20 K. No flavins were detected by e.p.r. spectroscopy. The cytochrome b-245 was not reduced by added NADH or NADPH. Dithionite-reduced cytochrome b-245 formed a complex with CO, supplied as a saturated solution, which was dissociated with 26 microseconds illumination from a xenon flash lamp, and the recombination with CO had a half-time of approx. 6 ms. Partly (80%) reduced cytochrome b-245 was oxidized by added air-saturated buffer with a half-time faster than 1 s at 20 degrees C, a resolution limited by mixing time. These results are compatible with cytochrome b-245 acting as an oxidase.

Protein methylesterase and leukocyte chemotaxis

The involvement of protein carboxylmethylesterase in leukotaxis has been studied. The enzyme is localized in the cytosolic fraction of neutrophils. In intact cells the presence of a chemoattractant induces a rapid stimulation of enzymatic activity. An half-maximal stimulation of this activity is induced by a concentration (3 n M) of peptide attractant that is close to that (1 n M) producing an half-maximal chemotactic response. In addition, the presence of a specific antagonist of peptide attractants lowers the stimulated esterase response of neutrophils to these chemotactic factors. The role of another enzyme, protein carboxylmethylase, has been re-examined. In the initial stages (1 min) of chemotactic stimulation, a highly variable increase in the activity of this enzyme occurs, while over a longer period (30 min) there is an apparent decrease. The results are consistent with a role for an increased turnover of protein methylesters in leukotaxis. The reversible neutralization of charge that would accompany this turnover process may contribute to alterations of membrane events that occur in the earliest stages of leukotaxis.

Cyclic Oscillation of Blood Neutrophils in a Patient With Multiple Myeloma

Abstract A patient with multiple myeloma developed periodic blood neutropenia (periodicity of 15–25 days) after 3 yr of intermittent treatment with cytotoxic agents. Peaks of serum colony-stimulating activity (CSA) level coincided with valleys of blood neutrophils. Fraction of marrow neutrophils in the multiplicative pool was high during blood neutrophil valleys and low during neutrophil peaks. In contrast, the maturation storage pool exhibited the reverse pattern. An increased fraction of marrow neutrophilic cells in the multiplicative pool was in active proliferation during a blood neutrophil valley and a decreased fraction during a blood neutrophil peak. These findings suggest that the marrow granulopoiesis was regulated through CSA. The defect causing the periodicity was probably related to the reduced number of neutrophils in the marrow maturation storage pool, which in turn may be related to a reduced and/or defective granulocytic stem cell pool size consequent to the long-term administration of cytotoxic drugs and/or infiltration of the marrow by myeloma cells.

Cyclic oscillation of blood neutrophils in a patient with multiple myeloma

A patient with multiple myeloma developed periodic blood neutropenia (periodicity of 15-25 days) after 3 yr of intermittent treatment with cytotoxic agents. Peaks of serum colony-stimulating activity (CSA) level coincided with valleys of blood neutrophils. Fraction of marrow neutrophils in the multiplicative pool was high during blood neutrophil valleys and low during neutrophil peaks. In contrast, the maturation storage pool exhibited the reverse pattern. An increased fraction of marrow neutrophilic cells in the multiplicative pool was in active proliferation during a blood neutrophil valley and a decreased fraction during a blood neutrophil peak. These findings suggest that the marrow granulopoiesis was regulated through CSA. The defect causing the periodicity was probably related to the reduced number of neutrophils in the marrow maturation storage pool, which in turn may be related to a reduced and/or defective granulocytic stem cell pool size consequent to the long-term administration of cytotoxic drugs and/or infiltration of the marrow by myeloma cells.