PMN Membranes Research Articles

Trypanosoma cruzi: Parasite-induced release of lysosomal enzymes by human polymorphonuclear leukocytes

The release of β-glucuronidase and lysozyme from human polymorphonuclear leukocytes (PMN) engaged in phagocytosis and lysis of Trypanosoma cruzi epimastigotes was studied in the presence or absence of chagasic serum. Lysosomal enzyme release was enhanced when parasites were sensitized with serum from a chronic Chagas' patient, increased up to 3 hr of incubation at 28 C, and depended on the PMN:parasite ratio. The release of lysosomal enzymes was determined by the presence of 2 m M cyanide, 2 μ M azide, 3 m M amobarbital, and 1 m M phenylbutazone. These drugs inhibited the killing of sensitized T. cruzi by interfering with the oxidative microbicidal mechanisms of PMN without affecting the uptake of the parasites. Lysosomal enzyme release occurred in the presence of cyanide and azide, indicating that in these cases the enzymatic release was unrelated to the killing of the parasites. Amobarbital and phenylbutazone, which stabilize PMN membranes, inhibited the release of β-glucuronidase and lysozyme by PMN. The addition of 10 μg/ml of cytochalasin B inhibited the phagocytosis and killing of sensitized T. cruzi by PMN but increased the enzymatic release by effector cells. Since cytochalasin B did not affect the close contact between PMN and parasites, it appears that the enzymes released to the extracellular milieu were not toxic to noningested parasites. Furthermore, the lysosomal enzymes did not lyse bystander unsensitized parasites. Therefore, the release of lysosomal enzymes during the interaction of T. cruzi epimastigotes and PMN seems to be related to the triggering event of the phagocytic process and does not bear a cause-effect relationship with parasite death.

Guanine nucleotides modulate the binding affinity of the oligopeptide chemoattractant receptor on human polymorphonuclear leukocytes.

The oligopeptide chemoattractant receptor on human polymorphonuclear leukocyte (PMN) membranes exists in two affinity states. Since guanine nucleotides regulate the binding affinity and transductional activity of several other types of receptors, we examined the effect of nucleotides on the binding of N-formyl-methionyl peptides to their receptors on human PMN membranes. The addition of guanylylimidodiphosphate (0.1 mM), a nonhydrolyzable derivative of guanosine triphosphate (GTP), to PMN membrane preparations reduced the fraction of high-affinity receptors detected in equilibrium binding studies from 21.3 +/- 0.13 to 11.8 +/- 0.05% (P less than 0.03), without altering the binding affinities. Since the total number of receptors remained unchanged, the effect of guanylylimidodiphosphate was to convert a portion of the receptors from the high-affinity state to the low-affinity state. At the maximal concentration of guanine nucleotide tested, approximately 50% of the high-affinity sites were converted to low-affinity sites. The findings obtained by equilibrium binding were supported by kinetic studies since the dissociation of the radiolabeled oligopeptide chemoattractant N-formyl-methionyl-leucyl-[3H]phenylalanine from PMN membranes was accelerated in the presence of guanine nucleotide. The effect of guanine nucleotides was reversed upon washing, indicating that affinity conversion is bidirectional. The guanine nucleotide effects were greatest with nonhydrolyzable derivatives of GTP followed by GTP then guanosine diphosphate. Neither guanosine monophosphate nor any adenine nucleotide tested had an effect on receptor binding. These data suggest a role for guanine nucleotides in the regulation of stimulus-receptor coupling of chemoattractant receptors on human PMN.

Subcellular localization of hydrogen peroxide production in human polymorphonuclear leukocytes stimulated with lectins, phorbol myristate acetate, and digitonin: an electron microscopic study using CeCl3

The ultrastructural H2O2-producing site in human polymorphonuclear leukocytes (PMN) stimulated with soluble stimuli was studied using a CeCl3-technique. CeLlular aggregation and formation of small vacuoles were observed when PMN were stimulated with 100 microgram/ml concanavalin-A, 1 mg/ml phytohemagglutinin, or 100 microgram/ml wheat germ agglutinin for 10 min at 37 degrees C. Electron-dense deposits formed from the reaction of H2O2 and CeCl3 were observed on the contact surface of the plasma membrane of aggregated PMN stimulated with lectins. Treatment with 5 microgram/ml cytochalasin-B before lectin- stimulation induced an enhanced formation of vacuoles, degranuLation, rounding of the contour, cellular aggregation, and enhancement of the deposits. Phorbol myristate acetate (PMA; 100 ng/ml) induced strong leukocyte aggregation, the formation of multiple huge vacuoles, degranulation, and H2O2 production at almost all of the contact surface between adjoining PMN and between PMN and erythrocytes, mononuclear cells, or thrombocytes. In PMN stimulated with digitonin (B microgram/ml), vacuolar formation, degranulation, multiple projections on the surface, and H2O2 production on the whole surface membrane were demonstrated. It is shown that cellular aggregation and cell-to-cell contact have an important role in the induction of O2- production induced by lectins or PMA and that O2- production induced by the detergent is not dependent on leukocyte aggregation.

Subcellular Localization of Hydrogen Peroxide Production in Human Polymorphonuclear Leukocytes Stimulated With Lectins, Phorbol Myristate Acetate, and Digitonin: An Electron Microscopic Study Using CeCl3

AbstractThe ultrastructural H2O2-producing site in human polymorphonuclear leukocytes (PMN) stimulated with soluble stimuli was studied using a CeCI3-technique. Cellular aggregation and formation of small vacuoles were observed when PMN were stimulated with 100 µg/ml concanavalin-A, 1 mg/ml Phytohemagglutinin, or 100 µg/ml wheat germ agglutinin for 10 min at 37°C. Electron-dense deposits formed from the reaction of H2O2 and CeCI3 were observed on the contact surface of the plasma membrane of aggregated PMN stimulated with lectins. Treatment with 5 µg/ml cytochalasin-B before lectin-stimulation induced an enhanced formation of vacuoles, degranulation, rounding of the contour, cellular aggregation, and enhancement of the deposits. Phorbol myristate acetate (PMA; 100 ng/ml) induced strong leukocyte aggregation, the formation of multiple huge vacuoles, degranulation, and H2O2 production at almost all of the contact surface between adjoining PMN and between PMN and erythrocytes, mononuclear cells, or thrombocytes. In PMN stimulated with digitonin (8 µg/ml), vacuolar formation, degranulation, multiple projections on the surface, and H2O2 production on the whole surface membrane were demonstrated. It is shown that cellular aggregation and cell-to-cell contact have an important role in the induction of O2– production induced by lectins or PMA and that O2– production induced by the detergent is not dependent on leukocyte aggregation.

Subcellular localization of H2O2 production in human neutrophils stimulated with particles and an effect of cytochalasin-B on the cells

The ultrastructural localization of H2O2 production in suspended polymorphonuclear leukocytes (PMN) stimulated with particles was studied using CeCl3 technique. PMN stimulated with opsonized zymosan or polystylene latex with or without IgG were incubated in 0.1 M Tris- maleate buffer with 1 mM CeCl3 and 10 mM aminotriazole. Cells were then fixed and embedded in a resin for electron microscopy. The reaction product of cerium perhydroxide was observed on the phagosomal membranes and on the areas of the plasma membrane engulfing the particles. Catalase or ferricytochrome-c decreased the deposits. p-Benzoquinone (O2- scavenger) inhibited the formation of the deposits, but KCN or NaN3 enhanced it. Pretreatment with p-diazobenzenesulfonic acid inhibited the reaction. In some PMN pretreated with cytochalasin-B, cellular aggregation was observed. The H2O2 production in these cells were observed on the membrane adherent to the particles and on the contact surface of the membrane of adjoining PMN. The plasma membrane was damaged and the electron-dense product was diffused into the cytoplasm. These results clearly show that H2O2 production is initiated at the area of the plasma membrane adherent to the particles and that H2O2 is released before the completion of phagocytosis.

Subcellular Localization of H2O2 Production in Human Neutrophils Stimulated With Particles and an Effect of Cytochalasin-B on the Cells

The ultrastructural localization of H2O2 production in suspended polymorphonuclear leukocytes (PMN) stimulated with particles was studied using CeCl3 technique. PMN stimulated with opsonized zymosan or polystylene latex with or without IgG were incubated in 0.1 M Tris-maleate buffer with 1 mM CeCl3 and 10 mM aminotriazole. Cells were then fixed and embedded in a resin for electron microscopy. The reaction product of cerium perhydroxide was observed on the phagosomal membranes and on the areas of the plasma membrane engulfing the particles. Catalase or ferricytochrome-c decreased the deposits. p-Benzoquinone (O2- scavenger) inhibited the formation of the deposits, but KCN or NaN3 enhanced it. Pretreatment with p-diazobenzenesulfonic acid inhibited the reaction. In some PMN pretreated with cytochalasin-B, cellular aggregation was observed. The H2O2 production in these cells were observed on the membrane adherent to the particles and on the contact surface of the membrane of adjoining PMN. The plasma membrane was damaged and the electron-dense product was diffused into the cytoplasm. These results clearly show that H2O2 production is initiated at the area of the plasma membrane adherent to the particles and that H2O2 is released before the completion of phagocytosis.

Membrane fluidity changes accompanying phagocytosis in normal and in chronic granulomatous disease polymorphonuclear leukocytes

We have studied membrane fluidity changes in polymorphonuclear leukocytes (PMN) during phagocytosis. Membrane fluidity was assessed by electron spin resonance (ESR) using a nitroxide-substituted stearic acid analog (5DS) as a spin probe. PMN from normal subjects and from 3 CGD patients (2 males, 1 female) were incubated in Kreb's Ringers phosphate with or without opsonized zymosan. ESR spectra were obtained and the order parameter (S), which is inversely related to membrane fluidity, was calculated. Without zymosan addition, S for normal (0.638) and for CGD (0.635) were not significantly different (p less than 0.35). The S values indicate that under resting conditions the molecular environment of the CGD membrane is similar to that of normal PMN membranes. However, with addition of opsonized zymosan, the normal, but not the CGD, PMN showed a significant increase (CGD, S = 0.638; normal, S = 0.647; p less than 0.001). This change in S for the normals is consistent with a more restricted movement of 5DS. Treatment of normal PMN with a mixture of scavengers specific for H2O2 (catalase, 1600 U/ml), O2-.(superoxide dismutase, 100 micrograms/ml), and for HO., (sodium benzoate, 1mM) during zymosan stimulation gave S values similar to those of resting cells. Catalase alone also lowered S value, suggesting that H2O2 was instrumental in causing the initial S value increase. This idea was supported by studies in which CGD cells were incubated with zymosan in the presence of glucose oxidase, an enzyme that catalyzes glucose oxidation resulting in the direct reduction of molecular oxygen to H2O2. Our results indicate that reduced O2 by- products, particularly H2O2, can cause altered biophysical properties of PMN membrane during phagocytosis.

Membrane Fluidity Changes Accompanying Phagocytosis in Normal and in Chronic Granulomatous Disease Polymorphonuclear Leukocytes

We have studied membrane fluidity changes in polymorphonuclear leukocytes (PMN) during phagocytosis. Membrane fluidity was assessed by electron spin resonance (ESR) using a nitroxide-substituted stearic acid analog (5DS) as a spin probe. PMN from normal subjects and from 3 CGD patients (2 males, 1 female) were incubated in Kreb's Ringers phosphate with or without opsonized zymosan. ESR spectra were obtained and the order parameter (S), which is inversely related to membrane fluidity, was calculated. Without zymosan addition, S for normal (0.638) and for CGD (0.635) were not significantly different (p less than 0.35). The S values indicate that under resting conditions the molecular environment of the CGD membrane is similar to that of normal PMN membranes. However, with addition of opsonized zymosan, the normal, but not the CGD, PMN showed a significant increase (CGD, S = 0.638; normal, S = 0.647; p less than 0.001). This change in S for the normals is consistent with a more restricted movement of 5DS. Treatment of normal PMN with a mixture of scavengers specific for H2O2 (catalase, 1600 U/ml), O2-.(superoxide dismutase, 100 micrograms/ml), and for HO., (sodium benzoate, 1mM) during zymosan stimulation gave S values similar to those of resting cells. Catalase alone also lowered S value, suggesting that H2O2 was instrumental in causing the initial S value increase. This idea was supported by studies in which CGD cells were incubated with zymosan in the presence of glucose oxidase, an enzyme that catalyzes glucose oxidation resulting in the direct reduction of molecular oxygen to H2O2. Our results indicate that reduced O2 by-products, particularly H2O2, can cause altered biophysical properties of PMN membrane during phagocytosis.

Characterization of lipoteichoic acid binding to polymorphonuclear leukocytes of human blood.

Human polymorphonuclear leukocytes (PMN) were shown to possess specific binding sites for lipoteichoic acid (LTA). LTA binding was reversible and time and temperature dependent. Scatchard plot analysis revealed an apparently single population of 6.6 X 10(6) LTA binding sites per PMN with a dissociation constant of 5.6 microM. Attachment of an avirulent, unencapsulated, M-negative strain of group A streptococci to PMN was inhibited by LTA, but not by other bacterial somatic antigens tested. Occupation of 30% of the LTA binding sites resulted in greater than 70% inhibition of streptococcal attachment to PMN. In contrast, LTA failed to block attachment of Escherichia coli or antibody-coated streptococci, indicating that binding sites for E. coli and the Fc portion of immunoglobulin G are distinct from those for LTA. Immunofluorescent studies demonstrated that LTA remained uniformly bound to PMN membranes for as long as 2 h at 37 degrees C. Cross-linking of PMN-bound LTA with anti-LTA resulted in rapid capping of LTA receptor sites. The results suggest that LTA is a monovalent ligand interacting with mobile receptors in the plasma membrane of PMN.

Orientation of the NADPH dependent superoxide generating oxidoreductase on the outer membrane of human PMN's

Summary The ratio of superoxide formation to O2 consumption in intact cell suspensions and subcellular particulate fractions rich in oxidoreductase activity, and the response seen upon adding NADPH and NADP+ to these fractions, suggest that the oxidoreductase is orientated in a trans configuration on the outer membrane. Its NADPH electron accepting site appears to lie on the cytosolic side of the outer membrane, whereas its superoxide generating site appears to lie on the opposite, extracellular side of the outer membrane.

Changing patterns of plasma membrane-associated filaments during the initial phases of polymorphonuclear leukocyte adherence.

By utilizing a combination of several ultrastructural techniques, we have been able to demonstrate differences in filament organization on the adherent plasma membranes of spreading and mobile PMN as well as within the extending lamellipodia. To follow the subplasmalemmal filaments of this small amoeboid cell during these kinetic events, we sheared off the upper portions of cells onto glass and carbon surfaces for 30 s--5 min. The exposed adherent membranes were immediately fixed and processed for high-resolution SEM or TEM. Whole cells were also examined by phase contrast microscopy, SEM, and oriented thin sections. Observed by SEM, the inner surface of nonadherent PMN membranes is free of filaments, but within 30 s of attachment to the substrate a three-dimensional, interlocking network of globular projections and radiating microfilaments--i.e., a subplasmalemmal filament complex--is consistently demonstrable (with or without postfixation in OsO4). Seen by TEM, extending lamellipodia contain a felt of filamentous and finely granular material, distinct from the golbule/filament complex of the adjacent adherent membrane. In the spread cell, this golbule-filament complex covers the entire lower membrane and increases in filament-density over the next 2--3 min. By 3--5 min after plating, as the PMN rounds up before the initiation of amoeboid movements, another pattern emerges--circumferential bands of anastomosing filament bundles in which thick, short filaments resembling myosin are found. This work provides structural evidence on the organization of polymerized contractile elements associated with the plasma membrane during cellular adherence.

Trypan Blue Inhibits Complement-Mediated Phagocytosis by Human Polymorphonuclear Leukocytes

Trypan blue completely inhibited attachment of human polymorphonuclear leukocytes (PMN) to Sepharose beads coated with C3 ant to sheep erythrocytes coated with IgM plus C3, but it did not inhibit attachment to erythrocytes coated with IgG. These results suggested that trypan blue inhibited C-mediated attachment to PMN membranes. Corroborative studies were performed with a strain of Staphylococcus aureus that requires C but not antibody, for opsonization and that activates the alternative pathway. Trypan blue was not toxic to PMN or bacteria, did nto interfere with immunoglobulin or C interactions, and did not affect attachment of opsonins to bacteria. However, the dye impaired PMN killing of S. aureus in normal nonimmune serum by inhibiting bacterial attachment to and ingestion by PMN. Further evidence that the inhibition was at the C3 receptor level came from the observations that, 1) once staphylococci were attached to PMN at either 37 degrees C or 0 degrees C, addition of trypan blue did not inhibit killing; and 2) trypan blue inhibited killing of bacteria opsonized with serum sufficient in C but previously absorbed at 0 degrees C with the same strain of organism to deplete specific antibody. Further studies with this agent may elucidate the roles of opsonic receptors on human phagocytes.

Neutrophilic leukocyte membrane proteins. I. Isolation.

Rabbit exudate-derived PMN were homogenized and the cell membranes isolated on a two-phase aqueous system. Glycoproteins were extracted from cell membranes with lithium diiodosalicylate. SDS polyacrylamide gel electrophoretic analysis showed a consistent pattern of three major glycoprotein entities. Cells radioiodinated supravitally showed most of the radioactivity associated with larger glycoprotein entities whereas PMN membranes radiolabeled after isolation yielded a single major peak of radioactivity associated with a much smaller protein entity. Heterologous antisera against rabbit PMN, PMN membranes, and membrane glycoproteins were all cytotoxic for PMN in the presence of complement, and all bound to the PMN surface as demonstrated with immunocolloidal gold on electron microscopy. The data suggest that one or more glycoprotein entities are membrane-associated ectoglycoproteins which can be radiolabeled supravitally.

Transport of sodium, potassium, and calcium across rabbit polymorphonuclear leukocyte membranes. Effect of chemotactic factor.

The transport properties of the rabbit peritoneal polymorphonuclear leukocyte (PMN) plasma membrane to Na+, K+, and Ca2+ have been characterized. The use of a silicone oil centrifugation technique provided a rapid and reliable method for measuring ion fluxes in these cells. Na+ and K+ movements across PMN membranes were found to be rapid. The value for the unifirectional steady-state fluxes (in meq/liter cell X min) were of the order of 3.0 for Na+ and 7.4 for K+. Ouabian inhibited both K+ influx and Na+ efflux, the latter being also dependent on the presence of extracellular potassium. The rate constant (in min-1) for 45Ca influx was found to be .05 and that for 45Ca efflux .04. The synthetic chemotactic factor formyl-methionyl-leucyl-phenylalanine (FMLP) was found to affect the fluxes of Na+, K+, and Ca2+ at concentrations as low as 10(-10)M. FMLP induced a large and rapid increase in the permeability of the PMN plasma membrane to 22Na. Smaller and delayed enhancements of 42K influx and 22Na efflux were also noted. Some evidence that the latter findings are a consequence of the increased 22Na influx is presented. 45Ca influx and efflux were also stimulated by FMLP. In the presence of 0.25 mM extracellular calcium, FMLP induced an increase in the steady-state level of cell-associated 45Ca. In the presence of .01 mM extracellular calcium, however, a transient decrease in the steady-state level of cell-associated 45Ca was induced by FMLP. The curves relating the concentration of FMLP to its effects on cation fluxes are very similar to those found for its enhancement of migration.

CELL ELASTIMETRY IN THE DETECTION OF IMMUNE NEUTROPENIA - - DEMONSTRATION OF A MEMBRANE PERTURBATION

Anti-neutrophil antibodies (ANA) have been demonstrated in the serums of recipients of multiple transfusions, mothers of infants with transient neonatal neutropenia, and in some patients with idiopathic neutropenia. Ingestion of sensitized neutrophils by other phagocytic cells has been observed, but direct evidence of membrane alteration of polymorphonuclear leukocytes (PMNS) by ANA is limited. We have used cell elastimetry to explore the problem. This technique measures, under direct visualization, the negative pressure required to aspirate PMNS into small pored pipettes. We have previously shown that the assay primarily reflects rigidity or deformability of the cell membrane. When incubated with normal PMNS, eight of nine serums from patients with known ANA significantly decreased membrane deformability -- i.e. cells became more rigid. The mean negative pressure required to aspirate PMNS incubated in normal plasma was 11.4 ± 3.6 (2 S.D.) cm Hg. PMNS incubated in the various ANA positive serums ranged from 25.9 to >43 cm Hg negative pressure required for aspiration. To insure objectivity, the study was conducted in an entirely blind fashion. Randomly coded serums from patients and controls were studied for deformability by observers unaware of the code. We conclude: 1) Elastimetry is a reliable and sensitive probe for the detection of ANA; 2) A primary effect of ANA upon PMN membranes has been demonstrated.

EFFECTS OF SURFACE ACTIVE AGENTS ON NEUTROPHIL RECEPTORS

An easily performed assay to identify the C3b and Fc receptor on human polymorphonuclear leukocytes (PMN) was developed. Salmonella typhi were directly fluoresceinated and then incubated in non-immune fresh human serum which led to C3b fixation via activation of the alternative pathway. Similarly, Type II pneumococci were fluoresceinated and opsonized with type specific rabbit anti-serum. PMN bearing C3b and Fc receptors formed rosettes with the respective bacteria which were easily readable because of their bright fluorescence. Studies employing various surface agents were employed to provide information on the physical properties of PMN receptors. Incubation of PMN at 37° C with C3-coated bacteria generated 54±5% C3b rosettes whereas PMN incubated with IgG coated bacteria yielded 78±8% rosettes. Heat inactivation of the fresh human serum at 56° C for 30 minutes completely inhibited the formation of the C3b and addition of goat anti-rabbit IgG inhibited the formation of the Fc rosettes. Pre-incubation of PMN with 1 mg/ml Trypsin, 1 mM N-ethyl maleimide, xanthine-xanthine oxidase to generate 20 nm superoxide anion and hydrogen peroxide (H2O2), 10 mM H2O2, and 8×10−4 M hydrocortisone reduced the number of C3b rosettes to values of 25±6%, 7±6%, 22±4%, 36±2%, and 24±5%, respectively, but had no significant effect on the number of Fc rosettes. Thus, this study provides evidence for selective receptors for C3b and Fc on PMN membranes.