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M S Klempner

Publications and source records attributed to M S Klempner.

At least 73 records · Page 4Linked to original sources
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

Tetanus toxin inhibits secretion of lysosomal contents from human macrophages.

The mechanism by which tetanus toxin (TT) inhibits the release of neurotransmitters from neurons is unknown. Since secretion of lysosomal contents from monocytes/macrophages (MOs) appears to be similar to the release of synaptosomal contents from neural cells, we examined the effects of TT on MO secretion. Adherent human MOs were cultured overnight with or without TT (1.25 micrograms/ml), washed, and resuspended in TT-free medium. In response to the calcium ionophore A23187, TT-treated MOs secreted significantly less lysozyme (27.2% +/- 3.2%) than did control cells (48.6% +/- 6.8%; 44.1% inhibition, P less than .001). TT inhibition of A23187-stimulated release of lysozyme from MOs was dose dependent, and higher concentrations of A23187 could not overcome the inhibitory effect of TT. MO secretions in response to a second secretagogue, phorbol myristate acetate, was also inhibited by TT. Heated TT (100 C for 1 hr), which has no in vivo toxicity, also failed to inhibit MO secretion. The inhibition of secretion by TT required holotoxin. C- and B-fragments produced no appreciable effect on secretion in response to either secretagogue. These studies indicate that human MOs may be a readily accessible, model system for the study of the biochemical basis of TT intoxication. However, observations in this system must be confirmed in neurons, the natural site of TT intoxication.

Calcimycin↗
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

Effects of leukocyte inhibitory factor (LIF) on human neutrophil function.

The effects of the lymphokine, leukocyte inhibitory factor (LIF), on human neutrophil function were studied. This soluble mediator, which is defined by its specific inhibition of neutrophil locomotion, does not interfere with chemotactic factor binding and does not affect basal or stimulated superoxide generation by neutrophils. In contrast, phagocytosis of opsonized Staphylococcus aureus is markedly inhibited by LIF, and degranulation is stimulated by this lymphokine. The possible mechanisms of LIF action on neutrophils are discussed.

Cells, Cultured↗
PubMed1983

Attachment of plasma membrane vesicles of human macrophages to Leishmania tropica promastigotes.

Intracellular parasitism of host macrophages is the pathologic hallmark of leishmaniasis. Since the organisms are found almost exclusively in this type of cell, the possibility that specific macrophage plasma-membrane determinants mediate the attachment to promastigotes of Leishmania tropica was investigated. Plasma membrane vesicles were prepared from human monocytes/macrophages and erythrocytes, radioiodinated, and incubated with L tropica promastigotes, erythrocytes, or Sephadex beads. Macrophage plasma-membrane vesicles bound to promastigotes to a significant extent but did not bind to intact erythrocytes or to an inert particle. In contrast, erythrocyte membrane vesicles did not bind to promastigotes. The binding of macrophage plasma-membrane vesicles to promastigotes demonstrated the characteristics of a receptor-ligand interaction in terms of specificity, saturability, competitive inhibition, and temperature independence. These results suggest the presence of one or more intrinsic binding sites on the macrophage plasma membrane to which promastigotes can attach. If this is the case, therapeutic intervention by strategies that inhibit attachment of this obligate intracellular parasite to its target host cell may be possible.

Animals↗
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↗
PubMed1982

Alterations in opsonophagocytic killing by neutrophils of Bacteroides fragilis associated with animal and laboratory passage: effect of capsular polysaccharide.

Electron microscopic studies of laboratory-passaged and animal-passaged isolates of Bacteroides fragilis showed significantly more capsular material around the latter. This observation correlated with increased survival of animal-passaged bacteria in rabbit intraperitoneal chambers and increased resistance to phagocytosis and opsonophagocytic killing by neutrophils. With an initial inoculum of 2.5 x 10(6) colony-forming units/ml, the number of bacteria surviving after incubation for 2 hr with neutrophils and pooled normal human serum was significantly (P less than 0.01) greater for animal-passaged than for laboratory-passaged bacteria. Neutrophil uptake of 14C-labeled animal-passaged and laboratory-passaged bacteria after incubation for 20 min was 45% and 63%, respectively (P less than 0.05). No significant difference in survival was found between animal-passaged and laboratory-passaged isolates of Bacteroides thetaiotaomicron. These findings suggest that on mechanism whereby the capsule of B. fragilis contributes to virulence is by inhibiting opsonophagocytic killing.

Animals↗
PubMed1982

Identification of a human neutrophil angiotension II-generating protease as cathepsin G.

A human neutrophil protease, initially termed neutral peptide-generating protease, has been shown to cleave angiotensin II directly from angiotensinogen and has been identified as leukocyte cathepsin G. When purified neutrophils were disrupted by nitrogen cavitation and fractionated by differential centrifugation, 44 and 24% of the angiotensin II-generating activity was in the lysosomal and undisrupted cell fractions, respectively. Cytochalasin B-treated human neutrophils stimulated with N-formyl-L-methionyl-L-leucyl-L-phenylalanine released beta-glucuronidase, lysozyme, and angiotensin II-generating protease in a dose-dependent fashion, consistent with localization of this protease to the neutrophil granule. Individually purified angiotensin II-generating protease and cathepsin G had similar proteolytic and esterolytic activity for angiotensinogen and N-benzoyl-L-tyrosine ethyl ester on a weight basis, exhibited identical mobilities by SDS-gradient polyacrylamide gel electrophoresis and pH 4.3 disc-gel electrophoresis, and gave precipitin lines of antigenic identity on Ouchterlony analysis with goat antibody to the angiotensin II-generating protease. Thus, the angiotensin II-generating protease of human neutrophils has been identified as cathepsin G on the basis of subcellular localization, substrate specificity, physicochemical characteristics, and antigenic identity.

Angiotensin II↗
PubMed1982

Specific binding of leukocyte inhibitory factor to neutrophil plasma membranes.

The interaction of leukocyte inhibitory factor (LIF), a lymphokine that acts as a cell-directed inhibitor of neutrophil locomotion, with the plasma membrane of its target cell was studied. LIF bound specifically to intact neutrophils and to neutrophil plasma membrane vesicles but not to intact macrophages, erythrocytes, or plasma membrane vesicles obtained from these cells. Although LIF adhered tightly to the neutrophil plasma membrane and was not removed by extensive washing, LIF activity could be eluted with N-acetyl-D-glucosamine. Elution with galactose, fucose, and N-acetyl-D-galactosamine did not remove LIF from the neutrophil plasma membrane. Similarly, N-acetyl-D-glucosamine, but not galactose, interfered with the binding of LIF to neutrophil plasma membrane vesicles. It is proposed that LIF binds to a high-affinity neutrophil plasma membrane site and that some intrinsic neutrophil locomotory abnormalities may represent tightly bound inhibitors of migration.

Absorption↗
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↗
PubMed1980

Neutrophil plasma membranes. I. High-yield purification of human neutrophil plasma membrane vesicles by nitrogen cavitation and differential centrifugation.

Neutrophil chemotaxis, phagocytosis, and oxygen-dependent microbicidal activity are initiated by interactions of stimuli with the plasma membrane. However, difficulties in neutrophil plasma membrane isolation have precluded studies on the precise structure or function of this cellular component. In this paper, a method is described for the isolation of representative human neutrophil plasma membrane vesicles, using nitrogen cavitation for cell disruption and a combination of differential centrifugation and equilibrium ultracentrifugation in Dextran gradients for membrane fractionation. Multiple biochemical markers and galactose oxidase-tritiated sodium borohydride surface labeling were employed to follow the yield, purity, and distribution of plasma membranes, nuclei, lysosomes, endoplasmic reticulum, mitochondria, and cytosol. According to these markers, neutrophil plasma membranes were exposed to minimal lysosomal hydrolytic enzymes and could be isolated free of other subcellular organelles. In contrast, disruption of neutrophils by mechanical homogenization resulted in > 20% lysosomal rupture and significant plasma membrane proteolysis. Electron microscopy demonstrated that plasma membranes isolated after nitrogen cavitation appeared to be sealed vesicles with striking homogeneity.

Cell Fractionation↗
PubMed1980

Disorders of phagocyte chemotaxis.

Recent advances in understanding the physiologic and biochemical bases for recruitment of phagocytes to inflammatory sites has led to the recognition of patients who have recurrent infections because of abnormalities of phagocyte chemotaxis. In some of these patients there is abnormal chemoattractant mediator production or regulation, whereas in others there are defects in phagocytic cell function. The cellular defects in chemotaxis can be characterized as either intrinsic defects of the cellular motility apparatus or acquired defects from mediators influencing cell function or from shifts in circulating phagocyte subpopulations. Systematic study of these defects has resulted in functional, biochemical, and ultrastructural characterization of abnormal phagocyte chemotaxis in certain patients, and in some patients study has led to rational approaches for treatment. Clinical trials assessing the efficacy of such pharmacologic agents are underway.

Adolescent↗
PubMed1980

The effect of hemodialysis and C5a des arg on neutrophil subpopulations.

Alterations in neutrophil subpopulations during human hemodialysis or following injection of C5a des arg into rabbits were studied. Whereas baseline peripheral blood neutrophils contained approximately 80% of cells that formed rosettes with IgG-sensitized erythrocytes, neutrophils harvested at the granulocyte nadir (20 min after initiating hemodialysis or the injection of C5a des arg) were markedly depleted of this population. This was seen in a change in ratio of rosette-forming neutrophils (RFN) to non-rosette-forming neutrophils (non-RFN) from 4:1 at 0 time to 1:2 at 20 min. Since non-RFN are less active in assays of adherence and chemotaxis, these alterations in circulating neutrophil populations were reflected in abnormal functional capacity of neutrophils harvested at 20 min. To study the mechanism of RFN depletion, we investigated the ability of C5a des arg to aggregate various human neutrophil suspensions. Unfractionated neutrophils and RFN demonstrated prompt in vitro aggregation in response to C5a des arg, whereas this activated complement fragment induced little aggregation in a population enriched for non-RFN. These results may explain the alterations in neutrophil adherence, chemotaxis, phagocytosis, and bactericidal activity, which have been reported to accompany clinical disorders characterized by in vivo complement activation (i.e., hemodialysis or gram-negative sepsis).

Animals↗