Differential augmentation by recombinant human tumor necrosis factor-alpha of neutrophil responses to particulate zymosan and glucan

Zymosan (Z) and its major insoluble carbohydrate component beta-linked glucan activate human neutrophils (PMN) through a trypsin-sensitive recognition mechanism. This mechanism is believed to involve the PMN CR3R. Both Z and glucan generated dose and time-dependent release of the secondary lysosomal granule marker vitamin B12 binding protein, leukotriene B4 (LTB4) and superoxide from PMN and were phagocytosed with similar dose-dependent kinetics. The PMN superoxide and LTB4 responses to glucan; however, were consistently greater than those to the same doses of Z. The phagocytosis of both particles was significantly reduced after partial digestion with beta-laminarinase but not beta-glucosidase or alpha-mannosidase suggesting a recognition mechanism dependent on intact beta-1,3-glucosidic bonds in both particles. TNF-alpha (rhTNF-alpha) promoted a time- and dose-dependent increase in the expression of PMN CR3 up to 60 min. The increased expression of CR3 was paralleled by the release of the secondary lysosomal granule marker vitamin B12-binding protein. This granule contains a population of CR3R in its boundary membrane and it is the fusion of this membrane with the plasma membrane that may represent the mechanism by which CR3 expression is increased. Preincubation of PMN with 10(-9)M rhTNF-alpha augmented phagocytosis, LTB4, and superoxide generation by PMN in response to activation by Z. In contrast, none of the responses to glucan was significantly increased after incubation with rhTNF-alpha. These differences suggest a lack of absolute homology between the recognition mechanisms for zymosan and glucan and that there is a component of the recognition mechanism for zymosan that is independent of that for glucan and is up-regulated after rhTNF-alpha pretreatment.     

Mechanisms of lysosomal enzyme release from human polymorphonuclear leukocytes. Effects of phorbol myristate acetate

PMA enhanced release of the azurophil granule enzyme, beta-glucuronidase, as well as lysozyme, from cytochalasin B-treated PMN's exposed to either zymosan particles or C5a. PMA was active at nanomolar concentrations, was not toxic to the cells, and was most effective when present for brief durations (0-1 min) before exposure of the cells to the stimuli. Beta-glucuronidase was not released in significant amounts from PMN's exposed to PMA alone, in the absence of stimuli such as zymosan or C5a. In contrast, only the specific granule enzyme, lysozyme, was released from unstimulated cells. Electron micrographs of cells exposed to PMA revealed an increase in the number of visible cytoplasmic microtubules as compared to control cells. Enhancement of lysosomal enzyme (beta-glucuronidase) release by PMA appears to be independent of effects on release of specific granule enzymes (lysozyme), but rather is likely due to PMA-induced elevations of cellular cGMP.     

Subcellular localization of the b-cytochrome component of the human neutrophil microbicidal oxidase: translocation during activation

We describe a new method for subcellular fractionation of human neutrophils. Neutrophils were disrupted by nitrogen cavitation and the nuclei removed by centrifugation. The postnuclear supernatant was applied on top of a discontinuous Percoll density gradient. Centrifugation for 15 min at 48,000 g resulted in complete separation of plasma membranes, azurophil granules, and specific granules. As determined by ultrastructure and the distribution of biochemical markers of these organelles, approximately 90% of the b-cytochrome in unstimulated cells was recovered from the band containing the specific granules and was shown to be in or tightly associated with the membrane. During stimulation of intact neutrophils with phorbol myristate acetate or the ionophore A23187, we observed translocation of 40-75% of the b-cytochrome to the plasma membrane. The extent of this translocation closely paralleled release of the specific granule marker, vitamin B12-binding protein. These data indicate that the b-cytochrome is in the membrane of the specific granules of unstimulated neutrophils and that stimulus-induced fusion of these granules with the plasma membrane results in a translocation of the cytochrome. Our observations provide a basis for the assembly of the microbicidal oxidase of the human neutrophil.     

Demonstration of plasma-membrane adenosine diphosphatase activity in rat lung

The adenosine diphosphatase (ADPase) activity of rat lung has been investigated. Subcellular fractionation of lung tissue homogenates by sucrose density gradient centrifugation has shown the ADPase activity to be associated with the plasma membrane. ADPase was solubilised from the membranes and fractionated by ammonium sulphate precipitation to separate a specific, low-Km ADPase from non-specific alkaline phosphatase activity. The solubilised ADPase has a Km of 50 microM at pH 7.5 and appears to be distinct from ATPase.     

Transmembrane potential changes during phagocytosis in rat alveolar macrophages

Studies were carried out to measure changes in the transmembrane potential of rat alveolar macrophages during exposure of the cells to zymosan particles or to the membrane perturbant, phorbol-12-myristate-13-acetate (PMA), and to determine if changes in membrane potential are related to superoxide anion release. Exposure of the cells to either zymosan or PMA leads to membrane depolarization, which precedes superoxide anion release. Furthermore, the magnitude of the depolarization is dependent upon the concentration of either zymosan or PMA. During exposure of the alveolar macrophages to increasing levels of zymosan, there is an increase in the amount of superoxide released as well as an increase in the magnitude of the depolarization. Incubation of the cells in medium containing 150 mM K+, a medium which causes membrane depolarization, leads to superoxide release from resting cells and a decrease in the amount of superoxide released from cells exposed to zymosan. These results indicate that release of superoxide anion from rat alveolar macrophages is related to membrane depolarization and suggest that the transmembrane potential change may act as a signal to initiate the phagocytotic responses of the cells.     

The fate of human polymorphonuclear leukocyte aminopeptidases upon cell stimulation with phagocytic and chemical stimuli.

1. After being treated with nonopsonized zymosan A or phorbol-12-myristate-13-acetate, human polymorphonuclear leukocytes release aminopeptidases together with granules marker-enzymes and vitamin B12-binding protein. 2. Chemotactic peptide, fMet-Leu-Phe and its analogs, fMet-Phe, fMet-Ala and fMet-Leu-Phe-Lys have a similar effect. 3. By their isoelectric point determinations the released aminopeptidases correspond to the enzymes from granules. Among aminopeptidases released the highest activities were those toward methionine- and alanine-2-naphthylamide and the lowest one toward arginine-2-naphthylamide.     

Human neutrophils contain subpopulations of specific granules exhibiting different sensitivities to changes in cytosolic free calcium

We examined the role of mobilization of intracellular calcium in the ability of human neutrophils to discharge specific granule constituents upon stimulation with the synthetic chemotactic factor, N-formyl-met-leu-phe. Extracellular calcium was not required for optimal secretion of the specific granule markers lactoferrin and vitamin B12-binding protein. Depletion and chelation of intracellular calcium, as well as reconstitution experiments, however, revealed different calcium requirements for stimulated secretion of these markers. N-formyl-met-leu-phe-induced secretion of vitamin B12-binding protein required half-maximal change in intracellular calcium of greater than 20 nM, while lactoferrin requirements were approximately 140 nM. Thus, it appears that cytosolic free calcium modulates fusion of subpopulations of specific granules which with the neutrophil plasma membrane.     

Intracellular organelle motility and membrane fusion processes in human neutrophils upon cell activation

Release and subcellular fractionation experiments indicate that fusion of a novel tertiary granule with the plasma membrane is concomitant with human neutrophil activation. Phorbol 12-myristate 13-acetate (PMA) induced a respiratory burst in human neutrophils as well as a high release of gelatinase, a marker of the tertiary granule. Preincubation of neutrophils with cytochalasin E induced a partially activated or 'primed' state, in which cells were unable to generate superoxide anion, but showed a reduced latency period for this activity. Fusion of tertiary granules with the cell surface also occurred during priming, although to a lesser extent than in PMA stimulation. The rapid tertiary granule degranulation, preceding that of specifics and azurophilics, seems to play an important role in the functionality and secretory properties of human neutrophils.     

Subcellular localization and properties of adenosine diphosphatase activity in human polymorphonuclear leukocytes

Adenosine diphosphatase (ADPase) activities were studied in human polymorphonuclear leukocytes with a recently developed radio-assay. The neutrophils were homogenized in isotonic sucrose and subjected to analytical subcellular fractionation. The sucrose density gradient fractions were assayed for ADPase activity and for principal organelle marker enzymes. ADPase activity was distributed between the plasma membrane, specific granule and soluble fractions. The plasma membrane and specific granule activities had similar kinetic and inhibitor properties but the cytosolic enzyme was clearly different. Studies with the non-penetrating inhibitor diazotized sulphanilic acid and measurements of latent activity indicate that plasma membrane ADPase activity is located on the external aspect to the cell. Its possible role in inhibiting platelet aggregation is discussed. Neutrophils were isolated from control subjects, patients with chronic granulocytic leukaemia and patients in the third trimester of pregnancy. The specific activities (mU/mg protein) of ADPase activity, in contrast to those of alkaline phosphatase, were similar in all three groups. This result, together with fractionation experiments and inhibition studies strongly suggests that ADPase activity is not attributable to neutrophil alkaline phosphatase.     

Modulation of the beta-adrenergic-response in cultured rat heart cells

Summary Subcellular fractionation studies in resting human neutrophils indicated a bimodal distribution for cytochrome b. A. major peak of cytochrome b co-sedimented with gelatinase under different experimental conditions. This localization was partially overlapped with specific granules (using lysozyme and lactoferrin as specific granule markers), but clearly resolved from azurophilic granules, plasma membrane, mitochondria, as well as from a novel alkaline phosphatase-rich intracellular organelle. A minor localization of cytochrome b was found in fractions enriched in both the plasma membrane marker 5-nucleotidase and alkaline phosphatase. A significant portion of ubiquinone cell content co-fractionated with the gelatinase-containing granules. After phorbol myristate acetate (PMA)-cell stimulation, cytochrome b was mobilized to fractions showing respiratory burst activity and enriched in 5-nucleotidase activity. This mobilization paralleled secretion of gelatinase and lysozyme to the extracellular medium. Furthermore, neutrophil stimulation with fluoride in the absence of cytochalasin B induced release of gelatinase and generation of superoxide anion with only minimal release of lysozyme. Preincubation of cells with the anion channel blocker 4,4-diisothiocyanostilbene-2,2-disulfonic acid (DIDS) prevented lysozyme release, but had only a minor effect on the release of gelatinase and did not inhibit the superoxide anion generation elicited by N-formyl-methionyl-leucyl-phenylalanine or PMA. These results suggest a main location of cytochrome b in mobilizable gelatinase-containing granules, which can constitute a subpopulation of specific granules. Furthermore, these findings show that the gelatinase-containing granule is functionally involved in the respiratory burst in neutrophils and that membrane fusion between plasma membrane and the gelatinase-containing granule occurs during activation of cells.Abbreviations DIDS 4,4-diisothiocyanostilbene-2,2-disulfonic acid - FMLP N-formyl-methionyl-leucyl-phenylalanine - PMA 4-phorbol, 12-myristate, 13-acetate     

Cytochrome b translocation to human neutrophil plasma membranes and superoxide release. Differential effects of N-formylmethionylleucylphenylalanine, phorbol myristate acetate, and A23187

The role of specific granules and cytochrome b in superoxide (O(2)) release was studied by comparing the effects of three different stimuli on normal human neutrophils, neutrophils congenitally deficient in specific granules, and granule-free normal neutrophil cytoplasts. Phorbol myristate acetate (PMA) stimulated normal neutrophils to release more O(2) than did N-formylmethionylleucylphenylalanine (fMet-Leu-Phe), which stimulated greater release than the calcium ionophore A23187. Neutrophils lacking specific granules produced variable amounts of O(2) in response to all stimuli. Stimulation with PMA, fMet-Leu-Phe, and A23187 produced maximal rates of O(2) release that were 32, 55, and 21% of that by normal cells. Likewise, granule-free neutrophil cytoplasts released 24, 20, and 0% of the O(2) released by intact cells. These data suggest that the stimuli require different mechanisms for activation. Three subcellular fractions (azurophil granule rich, specific granule rich, and plasma membrane rich) were separated by Percoll gradients from normal resting and stimulated neutrophils. In resting neutrophils, the cytochrome b content in the plasma membrane was 31% of the total, with the rest in the specific granule-rich fraction. Ten minutes after stimulation, PMA, fMet-Leu-Phe, and A23187 induced translocation of 27, 8, and 49%, respectively, of the cytochrome b from the specific granule-rich fraction to the plasma membrane. Although our data support a role for specific granule factors in A23187-induced O(2) release, there is no correlation between the amount of cytochrome b incorporated into the plasma membrane and the extent of O(2) production activated by the different stimuli.     

Platelet-activating factor as a stimulus of exocytosis in human neutrophils

The properties of platelet-activating factor (PAF-acether) 42–48ulus of exocytosis in human neutrophils have been re-investigated with particular attention to effects on cells that were not pretreated with cytochalasin B. Release of gelatinase, the most sensitive marker of exocytosis, was determined in addition to that of vitamin B-12-binding protein and β-glucuronidase. Superoxide production was assayed as a measure of the respiratory burst. The effects of PAF-acether were compared to those of leukotriene B₄ and N-formylmethionylleucylphenylalanine (fMet-Leu-Phe). Our results show that PAF-acether elicits marked secretion in untreated human neutrophils, and refute the prevalent view that cytochalasin B treatment is required for responsiveness. PAF-acether induced abundant release of gelatinase, increasing on average from 20% at 10 nM to 35% at 1 μM. This release was very rapid, i.e., almost complete after 2 min. fMet-Leu-Phe induced the same maximum response already at 0.1 μM, but release was considerably slower. Leukotriene B₄ was less potent with a maximum release of 20%. Exocytosis of gelatinase was always paralleled by liberation of smaller but significant amounts of vitamin B₁₂-binding protein from the specific granules. In contrast to their effect on exocytosis, PAF-acether and leukotriene B₄ were very weak stimuli of the respiratory burst when compared with fMet-Leu-Phe.     

Final digestion of starch in Musca domestica larvae. Distribution and properties of midgut α-d-glucosidases and glucoamylase

There are low-Mr (72,700) and high-Mr (330,000) soluble α-glucosidase activities in the hind-midgut of Musca domestica that can be isolated by ultracentrifugation. The low-Mr α-glucosidase is less stable and less inhibited by Tris than is the high-Mr α-glucosidase, and occurs mainly in hind-midgut contents, whereas the high-Mr α-glucosidase is found only in hind-midgut cells. Subcellular fractionation of hind-midgut cells showed that the high-Mr α-glucosidase is associated mainly with brush-borders, from where it is set free by freezing and thawing. The low-Mr α-glucosidase is recovered chiefly in the soluble fraction of the cell. The data suggest that the high-Mr and the low-Mr α-glucosidase occur mainly tightly and loosely bound to the cell glycocalyx, respectively. Based on subcellular fractionation, ultracentrifugation, and thermal inactivation data, there is only one molecular species of α-glucosidase and glucoamylase, which are solubilized by Triton X-100 from hind-midgut cell microvillar membranes. The results suggest that starch digestion is accomplished stepwise by luminal amylase, then by membrane-bound glucoamylase, and finally by glycocalyx-associated α-glucosidase and membrane-bound α-glucosidase. Luminal α-glucosidase probably digests ingested oligomaltodextrins and, since it is significantly excreted, it may also be involved in extracorporeal digestion.     

Properties of interleukin-1 as a complete secretagogue for human neutrophils

Human monocyte-derived Interleukin-1 (IL-1) stimulated a concentration-dependent extracellular release of azurophil (myeloperoxidase) and specific (vitamin B12-binding protein) granule constituents from cytochalasin B-treated human neutrophils. The serine protease inhibitors, L-1-tosylamide-2-phenylethyl-chloromethyl ketone (TPCK) and N-alpha-p-tosyl-L-lysine-chloromethyl ketone (TPCK) as well as an inhibitor of thiol protease activity, p-hydroxymercuribenzoate (PHMB), suppressed granule enzyme release from neutrophils activated with IL-1. Cycloheximide, an inhibitor of protein synthesis, had no effect on IL-1-induced neutrophil degranulation. Neutrophils pretreated with IL-1 were rendered unresponsive to subsequent exposure to this stimulus. IL-1-elicited granule exocytosis appears to be stimulus specific in that N-formyl-methionyl-leucyl-phenylalanine (FMLP), 1-0-hexadecyl/octadecyl-2-0-acetyl-sn-glyceryl-3-phosphorycholine (AGEPC), and 5(S),12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid (LTB4) were capable of eliciting a secretory response from IL-1-pretreated cells.     

Glycoprotein synthesis in the adult rat pancreas: II. Characterization of Golgi-rich fractions

Golgi-rich fractions were prepared from homogenates of adult rat pancreas by discontinuous gradient centrifugation. These fractions were characterized by stacks of cisternae associated with large, irregular vesicles and were relatively free of rough microsomes, mitochondria, and zymogen granules. The Golgi-rich fractions contained 50% of the UDP-galactose: glycoprotein galactosyltransferase activity; the specific activity was 12-fold greater than the homogenate. Such fractions represented < 19% of thiamine pyrophosphatase, uridine diphosphatase, adenosine diphosphatase, and Mg²⁺-adenosine triphosphatase. Zymogen granules and the Golgi-rich fractions were extracted with 0.2 m NaHCO3, pH 8.2, and the membranes were isolated by centrifugation. The glycoprotein galactosyltransferase could not be detected in granule membranes, while the specific activity in Golgi membranes was 25-fold greater than the homogenate.At least 35 polypeptide species were detected in Golgi membranes by polyacrylamide gel electrophoresis in 1% sodium dodecylsulfate. These ranged in molecular weight from 12,000 to <160,000. There were only minor differences between Golgi membranes and smooth microsomal membrane. In contrast, zymogen granule membranes contained fewer polypeptides. A major polypeptide, which represented 30–40% of the granule membrane profile, accounted for less than 3% of the polypeptides of Golgi membranes or smooth microsomal membranes.     

Solubilised intrinsic factor receptor from pig ileum and its characteristics.

The vitamin B-12-intrinsic factor receptor was shown to be present in pig ileum and localized on the brush borders of the enterocytes, and to be solubilisable with Triton X-100. At neutral pH and in the presence of Ca2+ it bound the vitamin B-12-intrinsic factor but not the vitamin B-12-cobalophilin complex. The solubilised vitamin B-12-intrinsic factor-receptor complex consisted of two molecular species with clear Stokes radii (13.11 and 33.53 nm), sedimentation coefficients (15.1 and 45.1 S) and molecular weights (1 600 000 and 12 000 000). A third smaller macromolecule possibly also represented the receptor. Some receptor activity was present in extracts prepared with buffers lacking detergent. There was evidence that the receptor is a membrane lipoprotein from which the lipids reversibly dissociate. Free intrinsic factor also bound to the solubilized receptor and its vitamin B-12-binding site seemed not to be involved in the attachment to the receptor. A small portion of the vitamin B-12-intrinsic factor spontaneously dissociated from the receptor and nearly all dissociated in the presence of Na2-EDTA. TheStokes radius of the dissociated vitamin B-12-intrinsic factor complex was 0.17 nm smaller than before binding to the receptor. Intrinsic factor and cobalophilin were present in ileal extract and observations were made on their molecular characteristics. These proteins, polymers of the vitamin B-12-intrinsic factor complex and binding of vitamin B-12 and its protein complexes to detergent micelles may give spurious receptor-like effects which must be properly controlled.     

Solubilised intrinsic factor receptor from pig ileum and its characteristics

The vitamin B-12-intrinsic factor receptor was shown to be present in pig ileum and localized on the brush borders of the enterocytes, and to be solubilisable with Triton X-100. At neutral pH and in the presence of Ca²⁺ it bound the vitamin B-12-intrinsic factor but not the vitamin B-12-cobalophilin complex. The solubilised vitamin B-12-intrinsic factor-receptor complex consisted of two molecular species with clear Stokes radii (13.11 and 33.53 nm), sedimentation coefficients (15.1 and 45.1 S) and molecular weights (1 600 000 and 12 000 000). A third smaller macromolecule possibly also represented the receptor. Some receptor activity was present in extracts prepared with buffers lacking detergent. There was evidence that the receptor is a membrane lipoprotein from which the lipids reversibly dissociate. Free intrinsic factor also bound to the solubilized receptor and its vitamin B-12-binding site seemed not to be involved in the attachment to the receptor. A small portion of the vitamin B-12-intrinsic factor spontaneously dissociated from the receptor and nearly all dissociated in the presence of Na₂-EDTA. The Stokes radius of the dissociated vitamin B-12-intrinsic factor complex was 0.17 nm smaller than before binding to the receptor. Intrinsic factor and cobalophilin were present in ileal extracts and observations were made on their molecular characteristics. These proteins, polymers of the vitamin B-12-intrinsic factor complex and binding of vitamin B-12 and its protein complexes to detergent micelles may give spurious receptor-like effects which must be properly controlled.     

Release of unsaturated vitamin B12 binding capacity from human granulocytes by the calcium ionophore A23187

The ionophore A23187, in the presence of calcium ions, was capable of eliciting the release of granule-associated unsaturated vitamin B₁₂ binding capacity from human granulocytes. The ionophore-induced extrusion of unsaturated vitamin B₁₂ binding capacity was concentration, time and temperature-dependent. The release was blocked by 2-deoxyglucose and was unaccompanied by cytotoxicity (trypan-blue uptake and lactate dehydrogenase release). The unsaturated vitamin B₁₂ binding capacity release was accompanied by the release of lysozyme, a specific granule marker enzyme.     

Activation of the oxygen-radical-generating system in granules of intact human neutrophils by a calcium ionophore (ionomycin).

Subcellular fractionation studies were performed on human neutrophils stimulated with ionomycin (a Ca(2+)-specific ionophore). The results of these studies revealed NADPH-oxidase activity, without any additive, both in the plasma membrane and in the specific granule fractions. After comparing these results with the NADPH oxidase activity induced by the ionophore in intact neutrophils, in differentiated HL-60 cells and in neutrophil cytoplasts, we conclude that ionomycin preferentially activates the NADPH oxidase pool located in the membrane of specific granules. Furthermore, we suggest that incorporation of granule membrane into the plasma membrane makes the associated NADPH oxidase less sensitive to activation induced by a rise in [Ca(2+)]i.     

Neutrophil migration across monolayers of resting or cytokine-activated endothelial cells. Role of intracellular calcium changes and fusion of specific granules with the plasma membrane.

Chemoattractants, used at concentrations to invoke optimal neutrophil chemotaxis, induce rapid changes in neutrophils such as a transient increase in intracellular Ca2+ ([Ca2+]i). We have previously observed that neutrophils adhering to cytokine-activated endothelial cells (EC) also respond with a rapid rise in [Ca2+]i caused by an endothelial membrane-bound form of platelet-activating factor. After preloading with the intracellular Ca(2+)-chelator bis-(O-aminophenoxyl)ethane-N,N,N',N'-tetraacetic acid (BAPTA/AM), neutrophils were no longer able to respond with a rapid rise in [Ca2+]i toward the chemoattractant FMLP or to rIL-1 beta-pretreated EC. These neutrophils were still able to adhere and migrate under the conditions tested. The only difference was that the BAPTA/AM-treated neutrophils migrated a little slower than untreated control neutrophils. This discrepancy was not observed at later time points. The BAPTA/AM-preloaded neutrophils did not differ from unloaded neutrophils in actin polymerization responses. Whereas untreated neutrophils demonstrated an up-regulation of the specific granule markers CD11b, CD45, and CD67 during migration (without any release from the azurophil granules), the BAPTA/AM pretreatment completely prevented this process. The BAPTA/AM-preloaded neutrophils did not release vitamin B12-binding protein from the specific granules upon treatment with FMLP. The down-modulation of the selectin member LAM-1, as seen upon neutrophil activation, was not affected by BAPTA/AM pretreatment of the neutrophils. Thus, neither the rapid rise in [Ca2+]i nor specific granule fusion with the plasma membrane constitute a prerequisite for neutrophil migration across resting or cytokine-activated EC.