Search PubMed⌕ Search

Biomedical subjects

I Spilberg

Publications and source records attributed to I Spilberg.

At least 37 records · Page 2Linked to original sources
PubMed1982

Sodium and potassium fluxes and membrane potential of human neutrophils: evidence for an electrogenic sodium pump.

Sodium and potassium ion contents and fluxes of isolated resting human peripheral polymorphonuclear leukocytes were measured. In cells kept at 37 degrees C, [Na]i was 25 mM and [K]i was 120 mM; both ions were completely exchangeable with extracellular isotopes. One-way Na and K fluxes, measured with 22Na and 42K, were all approximately 0.9 meq/liter cell water . min. Ouabain had no effect on Na influx or K efflux, but inhibited 95 +/- 7% of Na efflux and 63% of K influx. Cells kept at 0 degree C gained sodium in exchange for potassium ([Na]i nearly tripled in 3 h); upon rewarming, ouabain-sensitive K influx into such cells was strongly enhanced. External K stimulated Na efflux (Km approximately 1.5 mM in 140-mM Na medium). The PNa/PK permeability ratio, estimated from ouabain insensitive fluxes, was 0.10. Valinomycin (1 microM) approximately doubled PK. Membrane potential (Vm) was estimated using the potentiometric indicator diS-C3(5); calibration was based on the assumption of constant-field behavior. External K, but not Cl, affected Vm. Ouabain caused a depolarization whose magnitude dependent on [Na]i. Sodium-depleted cells became hyperpolarized when exposed to the neutral exchange carrier monensin; this hyperpolarization was abolished by ouabain. We conclude that the sodium pump of human peripheral neutrophils is electrogenic, and that the size of the pump-induced hyperpolarization is consistent with the membrane conductance (3.7-4.0 microseconds/cm2) computed from the individual K and Na conductances.

Biological Transport, Active↗
PubMed1982

Induction of a chemotactic factor from human neutrophils by diverse crystals.

The purpose of this study was to isolate and compare the chemotactic factor generated by human neutrophils after phagocytosis of three structurally different crystals. An apparently identical chemotactic factor for neutrophils was isolated from the granular fraction of human neutrophils allowed to phagocytose MSU, CPPD, or amorphous DC. The isolated chemotactic factors migrated identically on SDS polyacrylamide gel electrophoresis, with a relative motility of 8.05, and competed with MSU-induced chemotactic factor for binding sites on the human neutrophils in an identical fashion. The data support the concept that the generation of the chemotactic factor represents a general response of the neutrophil to the phagocytosis of foreign particles.

Binding, Competitive↗
PubMed1981

Demonstration of calcium-dependent chemotactic factor activatable esterase activity in human neutrophils: relationship with chemotaxis and chemotactic deactivation.

An increase in esterolytic activity, as measured by [3H] BAEE hydrolysis, was demonstrated in human neutrophil suspensions following incubation with three distinct chemotactic factors. The increased activity was demonstrated in intact cells as well as in cell lysates. The FMLP-enhanced enzymatic hydrolysis of BAEE was found to be calcium dependent. Esterase activation by FMLP was maximal at 1.0 mM of added calcium. Similar dose-response curves for esterase activation, chemotaxis, and chemotactic deactivation were obtained with the chemotactic factor FMLP, suggesting that in human neutrophils all three functions utilize some of the same early molecular events following chemotactic factor binding to th neutrophil surface.

Arginine↗
PubMed1981

Chemotactic factor receptor modulation and cytoskeletal structures.

The microfilament-disrupting agents cytochalasins A, B, and D were shown to impair the binding of the chemotactic factors [3H]formylmethionylleucylphenylalanine and 125 I-labeled crystal-induced chemotactic factor to their neutrophil receptor. Scatchard plot analysis revealed a decrease of the available binding sites in the cytochalasin B-treated cells. Cytochalasin B showed the same inhibitory profile on intact cells and on a membrane-rich preparation, suggesting that the effect was not dependent on an intact microfilament apparatus. The microtubule modifiers colchicine and vinblastine had no effect on the binding of the chemotactic factor to its cell receptors

Binding Sites↗
PubMed1981

Determination of a specific receptor for formyl-methionyl-leucyl-phenylalanine on th pulmonary alveolar macrophage and its relationship to chemotaxis and superoxide production.

3H-FMLP, a chemotactic peptide that resembles Escherichia coli chemotactic factor, is chemotactic for PAM, binds specifically to a site on the cell, and induces the generation of superoxide radicals by the cell. Scatchard analysis revealed an equilibrium dissociation constant at 26 degrees C of 1.45 x 10(-8)M and the presence of 1.7 , 10(5) receptors per cell. Binding was not inhibited by a partially purified C5a preparation or by the neutrophil-derived CCF but was inhibited by various N-formylated peptides. The order of potency of each peptide to inhibit 3H-FMLP binding was identical to the order of potency of each peptide to induce generation of superoxide by the PAM. Only small amounts of beta-glucuronidase activity and no lysozyme were detected in the supernatant after incubation of the cells for 30 min with varying concentrations of FMLP.

Animals↗
PubMed1980

Alteration of polymorphonuclear leukocyte surface charge by endogenous and exogenous chemotactic factors.

We investigated the effects of chemotaxins on the surface charge of isolated human PMN. Chemotaxis was ascertained using Boyden chambers. Surface charge was calculated using data derived from cell mobility as measured in a cytophorometer. The electrophoretic mobility of cells exposed to all chemotactic agents studied was altered. Endotoxin-activated serum containing C5a, PMN lysosomal extracts from "resting" and from crystal-stimulated cells, and Gly-His-Gly, a synthetic tripeptide, all significantly reduced the net negative surface charge of isolated neutrophils. Only chemotactically active substances effected this change; controls including heat-inactivated serum, other subcellular fractions, and an inert tripeptide, Gly-Gly-Gly, did not alter cell mobility in an electric field. These findings are consistent with studies by others on the effects of chemotactic factors on cation fluxes and cell aggregation and suggest a possible mechanism by which PMN directional migration is regulated.

Chemotactic Factors↗
PubMed1980

Stimulus-specific deactivation of chemotactic factor-induced cyclic AMP response and superoxide generation by human neutrophils.

The responses of isolated human peripheral neutrophils to either simultaneous or sequential additions of two chemotactic factors were studied. Simultaneous additions of formyl-methionyl-leucyl-phenylalanine (10-100 nM) and the fifth component of complement, C5a (1-10 microliters/ml), evoked partially additive responses of membrane depolarization as measured by the fluorescent dye 3,3'-dipropyl-thiocarbocyanine, a transient elevation of intracellular cyclic AMP (cAMP), and superoxide (O2-) generation as assessed by ferricytochrome c reduction. Preincubation of the cells with either formyl-methionyl-leucyl-phenylalanine or C5a alone caused dose-dependent inhibition of the depolarization, the cAMP increase, and O2- release induced by a subsequent exposure to an optimal dose of the same stimulus, i.e., deactivation occurred. In contrast, when cells were treated with one chemotactic factor and then exposed to the other stimulus, the cells exhibited a normal response of peak depolarization, the rise in cAMP, and O2-0 production i.e., cross-deactivation failed to occur. The results imply that deactivation of these phenomena is stimulus specific. Further, these observations are consistent with the hypothesis that cross-deactivation of chemotaxis is mediated by one or more processes that are irrelevant to O2- generation, and that occur distal to the depolarization and cAMP steps in the sequence of neutrophil activation: possibly microtubule polymerization and orientation.

Anaphylatoxins↗
PubMed1980

Superoxide generation and granule enzyme release induced by ionophore A23187. Studies on the early events of neutrophil activation.

The responses of isolated human peripheral neutrophils to activation by the divalent cation ionophore A23187 (4 x 10(-8) M to 2 x 10(-5) M) were studied over the course of 15 min at 37 degrees C. Dose-dependent increases were noted in the following A23187-induced functions: (1) membrane depolarization, as assessed by a fluorescent cyanine dye, (2) altered membrane permeability to 22Na+ and 45Ca2+, (3) increased intracellular levels of cAMP, (4) O2-generation, and (5) granule enzyme release. Membrane depolarization and the cAMP response preceded the onset of O2- generation and exocytosis, which began after a latency period of approximately 30 sec. All five functions induced by 2 x 10(-5) M A23187 required approximately 1 mM Ca0 for optimal expression and declined substantially as Ca0 was reduced towards a nominal O mM. In contrast, the Na+ ionophore gramicidin D (0.1 to 10 microgram/ml) caused cell depolarization and increased 22Na+ ionophore gramicidin D (0.1 to 10 microgram/ml) caused cell depolarization and increased 22Na+ uptake but did not stimulate 45Ca2+ uptake, the cAMP response, O2- generation, or exocytosis of granule constituents. The observations dissociate membrane depolarization and altered Na+ permeability, on the one hand, from a Ca2+ influx, cAMP response, O2- release, and exocytosis on the other. These studies, then, are consistent with a hypothesis that a Ca2+ influx is required for the biologic expressions of cell activation.

Anti-Bacterial Agents↗
PubMed1979

Chemotactic factor-induced generation of superoxide radicals by human neutrophils: effect of metabolic inhibitors and antiinflammatory drugs.

Human peripheral neutrophils generated superoxide radicals as assessed by ferricytochrome C reduction in response to activation by the synthetic chemotactic factor, N-formyl-methionyl-leucyl-phenylalanine. Superoxide generation was inhibited by 2-deoxy-D-glucose (ID50 4 X 10(-5)M), 2-iodoacetate (ID50 5 X 10(-5)M), and N-ethyl-maleimide (ID50 5 X 10(-6)M), suggesting a dependence on anaerobic glycolysis and sulfhydryl groups. Ouabain, microtubule-disrupting agents, inhibitors of respiration, oxidative phosphorylation, and protein and nucleic acid synthesis were without appreciable effects. Indomethacin (ID50 1 X 10(-4)M), ibuprofen (ID50 9 X 10(-4)M, and phenylbutazone (ID50 1 X 10(-5)M) all caused dose-dependent inhibition of superoxide generation at concentrations approximating those plasma and tissue levels obtained in human beings at therapeutic doses. Acetylsalicylic acid (125-500) microgram/ml) and aurothioglucose (10(-3)-10(-6)M) were without appreciable effects. Superoxide generation was inhibited only by relatively high concentrations of hydrocortisone (ID50 greater than 10(-3)M). Because superoxide radicals have been implicated in the pathogenesis of tissue injury in several forms of inflammation and arthritis in vivo, these studies suggest that the production of a potential cytotoxic factor may be subject to pharmacologic manipulation and that at least some of the antiphlogistic effects of the nonsteroidal antiinflammatory agents may be mediated through effects on superoxide production.

6-Aminonicotinamide↗
PubMed1979

Demonstration of a specific neutrophil receptor for a cell-derived chemotactic factor.

The crystal-induced chemotactic factor, a cell-derived chemoattractant for neutrophils, binds specifically to a site on human neutrophils but not to erythrocytes or lymphocytes, suggesting a relationship between the presence of specific binding sites on the neutrophils and the ability to be chemotactically activated. The Scatchard analysis revealed an equilibrium dissociation constant at 37 degrees C of 0.446 micrometer and the presence of approximately equal to 6.44 x 10(5) binding sites for 125I-crystal-induced chemotactic factor per cell. Binding was not displaced by the synthetic chemotactic factors F-Met-Leu-Phe and Gly-His-Gly or by complement-activated plasma providing evidence of the specificity of the receptor.

Binding Sites↗
PubMed1979

Mechanism of action of colchicine in acute urate crystal-induced arthritis.

Phagocytosis of urate crystals by human or rabbit neutrophils induces the synthesis and release of a glycoprotein, the crystal-induced chemotactic factor (CCF), which is chemotactically active both in vitro and in vivo. It has been proposed that CCF is a prime mediator of the acute gouty attack. Colchicine has been shown to decrease the production and release of this factor in vitro. In these studies, colchicine, at nonleukopenic doses, is shown to abrogate the acute arthritis induced by monosodium urate crystals in rabbits, but to have no effect upon the arthritis induced by the injection of the purified cell-derived chemotactic factor. Serum colchicine levels were 0.48-0.58 muM at 30 min and 0.12-0.3 muM at 90 min after intravenous injection of 0.2 mg/kg colchicine. Peripheral blood polymorphonuclear leukocytes obtained from colchicine-treated animals migrated normally towards a chemotactic stimulus but failed to produce CCF after phagocytosis of monosodium urate crystals. The dialyzed synovial fluid from rabbits injected with microcrystalline sodium urate contained chemotactic activity that was not present when animals were also given intravenous colchicine or injected intra-articularly with the chemotactic factor formyl-methionyl-leucyl-phenylalanine. Furthermore, the synovial fluid from rabbits injected with microcrystalline sodium urate significantly decreased (125)I-CCF binding to neutrophils. The binding of (125)I-CCF to its neutrophil receptor was not significantly reduced by the synovial fluid of colchicine-treated rabbits nor by the synovial fluid of control rabbits injected with the chemotactic factor formyl-methionyl-leucyl-phenylalanine. Colchicine (10 and 0.1 muM) was shown to have no effect upon the binding of (125)I-CCF to its cell receptor.

Animals↗
PubMed1979

Generation of superoxide radicals by human peripheral neutrophils activated by chemotactic factor. Evidence for the role of calcium.

In response to activation by the synthetic chemotactic factor FMLP, human peripheral neutrophils generated superoxide radicals as assessed by ferricytochrome C reduction. A dose-dependent increase in the amount of superoxide induced by FMLP over the concentration range of 1 X 10(-8) M to 1.6 X 10(-7) M was observed. Examination of the kinetics of the response revealed large amounts of superoxide generated by 1 min of incubation at 37 degrees C at an optimal dose of FMLP and a plateau effect after 5 min of incubation. Divalent cations did not influence the binding of 3H-FMLP to the cell, but superoxide generation by FMLP-activated neutrophils was observed to be dependent on the presence of divalent cations in the medium. In the absence of Mg2+, increasing Ca2+ ion concentration in the medium led to progressive increases in superoxide generation up to 4 mM, after which the response declined slightly. Mg2+, 0.25 to 4 mM, increased FMLP-induced superoxide generation to a much lower extent than did Ca2+. Lanthanum ion, 0.1 to 1 mM, in the presence of 1 mM Ca2+ inhibited the production of superoxide by FMLP 4 X 10(-8 ) M. Over the concentration range 3.3 X 10(-5 M to 3 X 10(-4 M, verapamil, a drug which selectively blocks the calcium channel, caused a dose-dependent inhibition of superoxide production and calcium-45 uptake in response to FMLP. This effect of verapamil could be overcome by increasing the concentration of Ca2+ in the medium. These observations suggest that a calcium influx plays an important role in the superoxide-generating capacity of the neutrophil.

Calcium↗
PubMed1979

Evidence for the role of superoxide radicals in neutrophil-mediated cytotoxicity.

Human peripheral neutrophils became cytotoxic to chicken red blood cells (CRBC) in the presence of lectins as assessed by release of 51chromium from labelled target cells. Phytohaemagglutinin (PHA) and concanavalin A (Con A), which caused time-dependent and dose-dependent cytotoxicity over a concentration range of 25--400 microgram/ml, also caused significant generation of superoxide radicals as measured by ferricytochrome C reduction. Pokeweed mitogen, which does not induce cytotoxicity over the same concentration range, was unable to promote superoxide generation by neutrophils. PHA-induced generation of superoxide paralleled and appeared to precede PHA-dependent cytotoxicity. Superoxide dismutase (SOD), which enzymatically destroys superoxide, caused moderate inhibition of PHA-dependent cytotoxicity over the concentration range of 100--500 microgram/ml whereas catalytically inactive enzyme had no effect. Incubation under oxygen-depleted conditions caused a marked decrease in both PHA-induced superoxide generation and cytotoxicity relative to that obtained with neutrophils incubated aerobically. These findings suggest a central role for superoxide radicals in causing target cell damage in this model of neutrophil-mediated cytotoxicity.

Catalase↗
PubMed1979

Chemotactic factor-induced superoxide radical generation by human neutrophils: requirement for proteinase (esterase) activity.

The requirement for proteinase (esterase) activity in the generation of O2- by human peripheral neutrophils was investigated. Neutrophils were activated by exposure to the chemotactic peptide FMLP and superoxide generation was assessed by ferricytochrome C reduction. Inhibition of O2- generation was observed by pretreating cells with the chloromethyl ketone derivatives of tosyl-phenylalanine (TPCK, ID50 2.2 X 10(-5)M) and tosyl-lysine (TLCK, ID50 1.9 X 10(-4)M). Dose-dependent inhibition was also noted with synthetic proteinase substrates, especially those of chymotrypsin-like specificity, phenylalanine and tryptophan methyl esters (ID50S approximately 1.5 X 10(-4)M), as compared to derivatives of basic amino acids, arginine and acetyl-lysine methyl esters, which caused negligible inhibition at 10(-3M. The inhibition of O2- generation demonstrated by TryME was time- and temperature-dependent and reversible with washing of the cells, whereas the inhibition seen with TPCK was irreversible. DFP at high concentrations, 10(-3)M or greater, caused moderate inhibition of O2- generation that was unaffected by exogenous serine and almost completely reversible by washing the cells. TPCK and TryME, at concentrations that caused marked inhibition of O2- generation, had no effect on the fmlp-induced calcium-45 uptake by the cells. These studies suggest that intact proteinase function is required for O2- generation and that this step follows the calcium influx in the activation sequence induced by FMLP.

Calcium↗