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B Styrt

Publications and source records attributed to B Styrt.

35 records · Page 2Linked to original sources
PubMed1988

An abnormal calcium uptake pump in Chediak-Higashi neutrophil lysosomes.

Calcium is mobilized from intracellular stores during phagocyte activation and appears to be involved in stimulus-response coupling in these and other cell types. Because the lysosome is a calcium-sequestering organelle in the neutrophil and abnormal lysosome morphology is associated with defective neutrophil function in Chediak-Higashi syndrome (CHS), we examined ATP-dependent calcium uptake in neutrophil lysosomes from the beige mouse model of CHS. We present findings indicating that CHS lysosomes have an enhanced capacity for ATP-dependent calcium uptake relative to control lysosomes. Kinetic analysis showed differences in Vmax and in the Km for both ATP and calcium, suggesting that both the number of lysosomal calcium uptake pumps and their substrate affinity may be altered in CHS. We conclude that a genetically determined abnormality of a subcellular calcium transport system may contribute to the structural and functional defects of CHS cells.

Adenosine Triphosphate↗
PubMed1988

Comparison of prostaglandins E2 and D2 as inhibitors of respiratory burst in neutrophils from atopic and nonatopic subjects.

Neutrophils from atopic and nonatopic donors were treated with prostaglandins D2 and E2 before stimulation of the respiratory burst. Both agents inhibited neutrophil response to formyl-methionyl-leucyl-phenylalanine, but superoxide production was inhibited much more profoundly by D2 than by E2. Inhibition was similar in atopics and nonatopics. Phorbol myristate acetate stimulation of superoxide production was not significantly altered by prostaglandins. These findings suggest that minor alterations in pathways of prostaglandin synthesis may have major effects on modulation of neutrophil function, and exploration of the mechanism of stimulus-specific inhibition may further elucidate the role of neutrophils in the inflammatory response.

Dinoprostone↗
PubMed1988

Characterization of the neutrophil respiratory burst in atopy.

Activated neutrophils may play a part in atopic disorders. In these studies, neutrophils were obtained from atopic and nonatopic adults for assessment of respiratory-burst activity. Superoxide production was measured in the resting state and after stimulation with phorbol myristate acetate, formyl-methionyl-leucyl-phenylalanine (f-met-leu-phe), or calcium ionophore A23187. Inhibition of this response by histamine was also measured. Neutrophils from atopic subjects produced more superoxide in the basal state, or in response to calcium ionophore A23187, or submaximal concentrations of f-met-leu-phe, than cells from control subjects. Histamine inhibition of f-met-leu-phe-stimulated superoxide production was less in atopic subjects than in control subjects. These findings suggest that neutrophils from atopic subjects may be hyperreactive in that they produce more superoxide in response to stimuli of low potency and are less readily inhibited than cells from control subjects. These properties may reflect a general attribute of cellular function in atopy and may contribute directly to tissue damage in allergic diseases.

Calcimycin↗
PubMed1988

Effects of pH on killing of Staphylococcus aureus and Escherichia coli by constituents of the neutrophil phagolysosome.

Lysosomotropic weak bases impair in-vitro neutrophil functions including intracellular killing of Staphylococcus aureus strain 502a. To investigate whether prevention of phagosomal acidification could account for impaired microbicidal activity, a model phagosome was formulated with a freeze-thawed granule extract as a source of lysosomal enzymes and H2O2 as a source of toxic oxygen metabolites. The lysosomal extract alone killed Escherichia coli strain S15 efficiently at pH 5.5 and 7.0, but had little activity against S. aureus 502a. Sublethal concentrations of the two agents, when combined, acted synergically against either organism. Each organism was killed more effectively at pH 5.5 than at pH 7.0 by the lysosome extract-H2O2 combination, but the killing of E. coli was more rapid than that of S. aureus in the same conditions. These findings suggest that impairment of neutrophil antistaphylococcal activity by weak bases may be mediated by their ability to raise phagosomal pH, and that persistence of E. coli in similar conditions does not occur because the latter is killed by lysosomal constituents in a non-pH-dependent fashion.

Adult↗
PubMed1987

Linkage between neutrophil degranulation and calcium discharge.

Calcium flux across organelle and plasma membranes is an important event in neutrophil activation. We measured calcium discharge into the media from neutrophils stimulated with formyl-methionyl-leucyl-phenylalanine after treatment with cytochalasin b. Cytochalasin markedly potentiated calcium efflux from stimulated neutrophils, and similarly promoted release of lysosomal enzymes into the media. Colchicine neither reproduced nor modified the cytochalasin effect. Neutrophil cytoplasts discharged very little calcium in response to stimulation, and discharge was not significantly altered by cytochalasin b. These findings indicate that neutrophil degranulation is accompanied by efflux of calcium into the media, and suggest that the neutrophil granules constitute a source of mobilizable calcium which could be used to modify the extracellular microenvironment.

Calcium↗
PubMed1986

Two functional domains in the phagocyte membrane glycoprotein Mo1 identified with monoclonal antibodies.

Granulocytes from patients genetically deficient in the leukocyte glycoprotein family, Mo1, LFA-1, and Leu-M5 (P150,94), have defective complement receptor type III (CR3) activity as well as abnormal adhesion-dependent functions such as spreading, chemotaxis, and phagocytosis. To determine the contribution of the Mo1 heterodimer deficiency to the various functional aberrations observed in deficient granulocytes, we mapped the functional domains of Mo1 using several monoclonal antibodies to this molecule. In addition to iC3b binding, two granulocyte adhesion functions were examined: Cell spreading on plastic coverslips and chemotaxis. One monoclonal antibody to Mo1, 44, inhibits all three functions. Other monoclonal antibodies (903, Leu-15, and OKM10) inhibit iC3b binding to granulocytes but have no effect on cell spreading and/or chemotaxis. Another antibody, 904, has no significant inhibition of iC3b binding but inhibits spreading on plastic and chemotaxis. These studies suggest the presence of two functional domains in Mo1: one involved in iC3b binding and the other in enhancing certain granulocyte adhesion-dependent functions.

Antibodies, Monoclonal↗
PubMed1986

Inhibition of neutrophil oxidative metabolism by lysosomotropic weak bases.

Maintenance of an acidic intralysosomal compartment may be relevant to multiple aspects of neutrophil function. The effect of lysosomal alkalinization on the neutrophil respiratory burst was studied by measuring cytochrome c reduction in response to soluble stimuli in the presence of lysosomotropic weak bases. The weak bases chloroquine, ammonium chloride, methylamine, and clindamycin all raised the intralysosomal pH and inhibited neutrophil oxidative metabolism at concentrations ranging from 0.1 to 100 mmol/L. Inhibition was dose dependent for each base and correlated significantly with the degree of lysosomal alkalinization. Concentrations that did not alkalinize the lysosome did not inhibit the respiratory burst. Inhibition by weak bases was seen when oxidative metabolism was stimulated by phorbol myristate acetate, calcium ionophore A23187, formyl-methionyl-leucyl-phenylalanine, opsonized zymosan, or sodium fluoride. Increasing the stimulus concentration (from 5 ng/mL to 5 micrograms/mL phorbol myristate acetate and from 0.5 to 1 mumol/L A23187) diminished or abolished inhibition by weak bases. Washing the cells after incubation with bases and before stimulation substantially reversed the inhibition. None of the bases impaired detection of superoxide in a cell-free xanthine-xanthine oxidase assay. Other indexes of oxidative metabolism, including oxygen consumption and hydrogen peroxide release, were also inhibited by weak bases. Analysis of particulate NADPH oxidase activity from neutrophils stimulated in the presence of bases suggested that these cells assemble a subnormal amount of an enzyme complex with normal kinetic characteristics. Lysosomotropic weak bases alkalinized the neutrophil lysosome and produced inhibition of oxidative metabolism that was dose related, was not stimulus specific, and was largely reversed by washing the cells before stimulation. A possible explanation would be altered assembly of the enzyme complex involved in respiratory burst activation as a consequence of impaired granule/plasma membrane fusion in the presence of diminished transmembrane pH gradients.

Ammonium Chloride↗
PubMed1985

Inhibition of neutrophil superoxide production by fanetizole.

The effects on neutrophil function of the new immunomodulatory agent fanetizole mesylate were studied. Fanetizole did not affect random or stimulated migration, phagocytosis, or degranulation by normal human neutrophils. Production of superoxide in response to the chemotactic factor formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe) was markedly inhibited (41.3 +/- 3.9%) by 250 microM fanetizole. This inhibition was not due to scavenging of superoxide by fanetizole, as there was no impairment of superoxide detection in a cell-free xanthine-xanthine oxidase system. Inhibition was dose dependent (no effect seen with 1 or 10 microM fanetizole) and stimulus specific (no impairment of superoxide production in response to phorbol myristate acetate). Washing the cells after fanetizole treatment partially restored their superoxide response to f-Met-Leu-Phe. Suppression of neutrophil production of toxic oxygen metabolites may partially explain the antiarthritic effect of fanetizole, and study of such selective inhibitors may be useful in probing the contribution of neutrophils to inflammatory tissue damage.

Adult↗
PubMed1985

Differential lysis of plasma membranes and granules of human neutrophils by digitonin.

The effect of the detergent digitonin on lysis of granule and plasma membranes of human neutrophils was studied. Either linear or sigmoid dose-response for release of the cytoplasmic marker lactate dehydrogenase and the granule markers lysozyme, beta-glucuronidase, lactoferrin, and myeloperoxidase was noted using digitonin concentrations ranging from 0.001 to 0.1 mM. However, release of the cytosol compartment was far more sensitive to the detergent than the granule compartment, with more release of lactate dehydrogenase than of lysozyme at 0.01-0.08 mM digitonin. Distinction between the two compartments was optimal at 0.025 mM digitonin. By examining in parallel the digitonin-induced release of exogenous fluorescent or luminescent indicators, a granule location was demonstrated for the pH indicator 9-aminoacridine, while the calcium probes aequorin and Quin 2 were released coincident with release of the cytosolic enzyme lactate dehydrogenase. These findings were employed to validate use of the indicators for monitoring of ion translocation in the intact cell. The differential effect of this detergent on subcellular membranes provides a broadly applicable technique for rapid assessment of the subcellular localization of tracer substances. Rapid monitoring may help to avoid problems of redistribution during cell fractionation.

Cell Fractionation↗
PubMed1985

Modification of interactions between neutrophils and staphylococci by lysosomotropic weak bases.

Weak bases that alkalinize the pH within neutrophil lysosomes inhibit in vitro cell functions, including lysosomal enzyme release and superoxide production. To determine the relevance of this inhibition to microbicidal activity, the effect of lysosomotropic weak bases on interactions between human neutrophils and Staphylococcus aureus 502a was studied. After treatment with 1 mM chloroquine, neutrophils showed significantly impaired phagocytosis of 14C-labeled S. aureus. However, 50 mM ammonium chloride had no effect on phagocytosis, although we have previously shown that this concentration raises lysosomal pH and inhibits degranulation and superoxide production. This base was therefore used to study effects on intracellular microbicidal activity. Incubation of neutrophils with 50 mM ammonium chloride diminished killing of S. aureus (22.9 +/- 6.3% of bacteria surviving versus 8.2 +/- 1.3% in suspensions without ammonium chloride). At 1 mM, ammonium chloride had no significant effect. The inhibition of cellular function could be neither explained as a function of neutrophil death, as measured by trypan blue dye exclusion, nor attributed to direct promotion of bacterial growth (in the absence of neutrophils, colony counts were similar in the presence or absence of ammonium chloride) or enhanced resistance to neutrophil microbicidal mechanisms (bacteria treated with ammonium chloride and washed before neutrophil exposure showed no improvement in survival). Ammonium chloride at 50 mM also impaired neutrophil killing of S. aureus in an anaerobic chamber, but microbicidal activity against Escherichia coli S15 was not affected. These findings suggest that optimal neutrophil killing of staphylococci requires a highly acid intralysosomal compartment, but ingestion of bacteria does not. This may reflect primary failure of acidification of the phagocytic vacuole or differential pH requirements for fusion of the plasma membrane with itself and with lysosome membranes. The difference between effects on killing of S. aureus and E. coli is probably a result of the relative importance of the components of neutrophil microbicidal activity against the two organisms.

Ammonium Chloride↗
PubMed1983

Alkalinization of the intralysosomal pH by clindamycin and its effects on neutrophil function.

Clindamycin is concentrated within human neutrophils and their lysosomes because the intralysosomal pH is highly acidic and this antibiotic behaves as a weak base. We have examined the ability of clindamycin to buffer (alkalinize) the intralysosomal pH and studied the consequences of alterations of lysosomal pH on neutrophil functions. At therapeutic concentrations of clindamycin (0.01 mM), we observed no effect on intralysosomal pH as monitored by the distribution and fluorescence of a weakly basic fluorescent probe 9-aminoacridine. However, at higher concentrations (1.0 mM), clindamycin alkalinized the intralysosomal pH and inhibited neutrophil lysosomal degranulation to several stimuli, and superoxide production in response to phorbol myristate acetate. Neutrophil locomotion was unaffected even at concentrations of clindamycin which increased the intralysosomal pH. We conclude that clindamycin accumulates in acidic neutrophil lysosomes and, at high concentrations, alkalinizes the intralysosomal pH. Coincident with lysosomal alkalinization there is inhibition of secretion and respiratory burst activity suggesting that intact lysosomal pH regulation is important for these functions. Clindamycin provides a useful tool to examine the relationships between weak base uptake, lysosomal pH and neutrophil functions.

Adult↗
PubMed1983

Alkalinizing the intralysosomal pH inhibits degranulation of human neutrophils.

Degranulation of lysosomes is one of the consequences of neutrophil activation. Regulatory mechanisms of lysosomal secretion are thought to be localized largely in the plasma membrane and cytosol, with the lysosome playing a passive role in secretion. Recent evidence indicates that the intralysosomal pH is highly acidic (pH congruent to 5.5) and is maintained by active transport of H+. We investigated whether changes in the intralysosomal pH altered the availability of lysosomes for exocytosis. Intralysosomal pH in intact neutrophils was monitored with the weakly basic fluorescent probe, 9-aminoacridine (9AA). The weak bases, methylamine, chloroquine, clindamycin, propanolol, and ammonium chloride (0.1-50 mM), caused an alkalinization of the intralysosomal pH as determined by reversal of quenching of 9AA fluorescence. Similarly, each of the weak bases, including ammonium chloride, methylamine, chloroquine, ethylamine, propylamine, propanolol, clindamycin, and dansylcadaverine, inhibited neutrophil degranulation in response to the calcium ionophore A23187, phorbol myristate acetate, or the chemotactic peptide, formyl-methionine-leucine-phenylalanine plus cytochalasin B. These studies indicate that an acid intralysosomal pH is important to the neutrophil secretory response and suggest that the lysosome may play an active part in control of degranulation.

Adult↗
PubMed1981

Clindamycin uptake by human neutrophils.

An anaerobic environment limits the microbicidal capacity of polymorphonuclear neutrophils (PMNs). To augment PMN killing under these conditions, the characteristics and mechanisms of clindamycin uptake by human PMNs were studied. The peak intracellular concentration of clindamycin was approximately 40 times greater than the extracellular concentration. Clindamycin uptake was rapid, saturable, and temperature-dependent. Intracellular accumulation of the drug was inhibited in acid pH, and agents that collapsed the transmembrane pH gradients also inhibited uptake. Isolated PMN lysosomes also accumulated clindamycin against a large concentration gradient, and uptake was reduced by collapsing the translysosomal membrane pH gradient. The intracellular drug was fully bioactive. These studies demonstrate that clindamycin is rapidly accumulated by PMNs, that drug uptake is related to a pH gradient, and that clindamycin appears to be lysosomotropic.

Carbonyl Cyanide m-Chlorophenyl Hydrazone↗
PubMedDate not supplied

Estrogens and infection.

The multiple effects of estrogens on infectious processes are only beginning to be understood. The existence of such effects is suggested by gender-related differences in the incidence and severity of some infections and by the association of certain infections with predictable hormonal changes. Current information indicates that estrogens may depress cell-mediated immunity, impair the activity of natural killer cells, and suppress some aspects of neutrophil function. Estrogens potentiate the production of systemic antibody, but local antibody responses may be impaired. Direct effects of estrogens on microorganisms have thus far been best studied in fungi; these hormones may either stimulate or suppress fungal virulence, depending on the species involved. Recent research also suggests responsiveness to estrogens in a wider variety of microorganisms. Studies in cell culture, animals, and humans indicate that pregnancy, estrogen supplementation, and menstrual stage can affect the acquisition and severity of certain bacterial, parasitic, and viral infections. This interaction depends on multiple attributes of both the microbe and the host in a given setting and thus may lead to disparate outcomes; however, there appears to be a predisposition to increased infectious morbidity in certain high-estrogen states. In view of the widespread use of estrogen supplementation, the clinical impact of estrogens on the incidence and outcome of infection needs to be better defined.

Antibody Formation↗