Jan G.R. Elferink

The role of cyclic nucleotides in neutrophil migration

1. The literature concerning the effects of cAMP and especially cGMP on neutrophil migration is reviewed. 2. Experiments with agents that enhance cGMP level, and with electroporated neutrophils in which cGMP was introduced, show that the nucleotide has different effects. There is a maximal stimulation at a specific concentration while higher concentrations are less effective or even inhibitory. 3. Some physiologically active peptides such as granulocyte-macrophage colony-stimulating factor (GM-CSF), atrial natriuretic factor, and endothelin appear to modify neutrophil migration via a cGMP-dependent mechanism. 4. Dependent on concentration and conditions (random migration vs. fMLP-activated migration, using nitric oxide (NO), NO donors, and inhibitors of NO synthase), NO has stimulatory or inhibitory effects on neutrophil migration. 5. The differential effects of cGMP and cAMP on neutrophil migration are discussed with regard to intracellular actions, metabolism, interaction with calcium, and relation to structural changes required for cell movement.

Thimerosal A versatile sulfhydryl reagent, calcium mobilizer, and cell function-modulating agent

An overview of the literature concerning the effects of thimerosal is presented. Because of its antibacterial effect, thimerosal is used for a variety of practical purposes such as antiseptic and preservative. In biomedical studies, thimerosal is used as a sulfhydryl reagent, and as a calcium-mobilizing agent. The ability of thimerosal to act as a sulfhydryl group is related to the presence of mercury. Relatively little study has been devoted to the mechanism of the reaction of thimerosal with the sulfhydryl group; the sulfhydryl reactive capacity is mostly concluded on the basis of inactivation of the effect by dithiothreitol (DTT). Thimersal causes a release of calcium from intracellular stores in many cells types; this is followed by an influx of extracellular calcium. Both InsP3- and ryanodine-sensitive calcium stores may be affected. Studies with permeabilized cells or organelles show that the effect of thimerosal on calcium is dependent on the concentration: low concentrations of thimerosal stimulate calcium release, high concentrations are inhibitory. This dependence is not found in intact cells. Thimerosal may activate or inhibit a number of cell functions. These are often related to the ability to release calcium or with the sulfhydryl reactivity. In platelets, thimerosal causes aggregation, increase of arachidonic acid metabolism, and exocytotic release of serotonin. In neutrophils, thimerosal causes, besides an increase of cytosolic free calcium, an increase of formyl-methionyl-leucyl-phenylalanine (fMLP)-activated leukotriene release, and a modulation of chemotactic migration and exocytosis. At low concentrations, thimerosal induces chemotactic migration of neutrophils, in the absence of other chemoattractants. The effect is also observed with thiosalicylic acid, indicating that the stimulation of migration was due to the thiosalicylic acid moiety of the thimerosal molecule. At higher concentrations, thimerosal causes inhibition of fMLP-activated migration. Low concentrations of thimerosal, but not of thiosalicylic acid, induced exocytotic enzyme release from neutrophils. High concentrations of thimerosal inhibited fMLP-activated exocytosis. The results point to an involvement of calcium mobilization and calcium influx of activation, and reaction with sulfhydryl groups for inhibition.

Chlorpromazine inhibits phagocytosis and exocytosis in rabbit polymorphonuclear leukocytes.

Abstract Phagocytosis of zymosan particles and the concomitant release of lysosomal enzymes by exocytosis in polymorphonuclear leukocytes is inhibited by chlorpromazine. Cytochalasin B prevents particle uptake, but not enzyme release; the divalent cation ionophore A 2318 7 induces enzyme release in the absence of zymosan if Ca2+ is present. In both cases enzyme release is inhibited by chlorpromazine, thus demonstrating an effect of chlorpromazine on exocytosis independent from its effect on phagocytosis. The results are discussed in relation to the role of Ca2+ in exocytosis and the effect of chlorpromazine on membrane properties.

Effect of interleukin 8 in meconium on in-vitro neutrophil chemotaxis

BACKGROUND Pneumonitis, characterised by large numbers of neutrophils in the lung, is an important feature of the meconium aspiration syndrome. The mechanism underlying the neutrophil influx is not known. We have investigated whether meconium has chemotactic activity and whether such activity is related to the presence of interleukin 8. METHODS The chemotactic activity of meconium on neutrophils from newborn infants was assessed in a Boyden-chamber assay. Interleukin 8 and formyl-methionyl-leucyl-phenylalanine (f-MLP) served as positive controls. Inhibition of chemotaxis was assessed with monoclonal antibody to interleukin 8. The interleukin-8 concentration was measured by ELISA. FINDINGS Sterile meconium suspension from seven unrelated newborn babies increased migration of neutrophils from neonates in comparison with random migration (79, 72, 70, 50, 58, 88 microm vs 46 microm; p<0.001). This effect was greatest at a meconium concentration of 5 g/L, although differences between samples from individual babies were observed. Interleukin 8 was present in all meconium suspensions (480-3980 ng/L). Anti-interleukin-8 inhibited neutrophil migration. INTERPRETATION Interleukin 8 is present in meconium and it induces chemotaxis of neutrophils in vitro. This mechanism may have a role in the pathogenesis of pneumonitis in meconium aspiration syndrome.

Endothelin-induced activation of neutrophil migration

Migration of rabbit peritoneal neutrophils was stimulated by endothelin-1 (ET-1) up to 2.10(-8) M. Higher concentrations inhibited random migration. The stimulating effect of ET-1 was inhibited by BQ-123, a specific antagonist of the ETA receptor. A checkerboard assay showed that the stimulating effect of ET-1 on neutrophil migration was chemokinetic rather than chemotactic. Extracellular Ca2+ was required for the activating effect of ET-1. Non-selective calcium channel blockers such as econazole and La3+ strongly inhibited ET-1-activated migration but had little effect on fMLP-activated migration, underlining the importance of Ca2+ influx for ET-1-activated migration. Studies with electroporated neutrophils showed that the increase in migration was most pronounced at calcium concentrations between 100 nM and 1 microM. ET-1-activated migration of electroporated cells was completely blocked by low concentrations of calcium-channel blockers such as verapamil and nitrendipine. Migration by intact cells was inhibited by the same concentration of verapamil, but to a lesser degree; nitrendipine had little effect on migration of intact cells. This suggests that calcium derived from intracellular stores is required for migration activated by ET-1. Protein kinase C, protein tyrosine kinase, and phosphatase activity were involved in the activating effect of ET-1 on neutrophil migration. ET-1 did not induce exocytotic enzyme release, in neither the presence nor the absence of cytochalasin B.

Toxicity of organotin compounds for polymorphonuclear leukocytes: The effect on phagocytosis and exocytosis

Phagocytosis and concomitant release of enzymes by rabbit polymorphonuclear leukocytes (PMNs) are inhibited by micromolar concentrations of triphenyltin and tributyltin; inhibition by triethyltin occurs at higher concentrations. Chemotactic peptide-induced exocytosis is inhibited at the same concentrations as phagocytosis. Tributyltin causes cell lysis at slightly higher concentrations as required for inhibition of phagocytosis and exocytosis. The organotin compounds have little effect on ATP level in PMNs, which makes an effect on metabolic energy providing processes unlikely. The increase of Ca2+-permeability of the plasma membrane, induced by chemotactic peptide, is inhibited by the organotin compounds. Inhibition of exocytosis by triphenyltin can be counteracted by a number of sulfhydryl compounds. The results suggest that the organotin compounds interfere with PMN function in an early phase of cell activation, where all functions have a common pathway, and where vulnerable sulfhydryl groups play a pivotal role.

Photodynamic treatment of yeast cells with the dye Toluidine blue : all-or-none loss of plasma membrane barrier properties

Photodynamic treatment of Kluyveromyces marxianus with the sensitizer Toluidine blue leads to the loss of colony forming capacity. In this paper, the influence of this treatment on the barrier properties of the plasma membrane has been studied. Photodynamic treatment with the dye Toluidine blue resulted in efflux of potassium ions and E260-absorbing material. Moreover, cells became stainable with erythrosine. It is concluded that the permeability change induced by photodynamic treatment proceeds in an all-or-none fashion. Treatment of this yeast strain, with the dye and light, also induced a diminution of the cell volume. This process is most likely not coupled to the cellular potassium content, but rather to the integrity of the vacuole. These data suggest that the vacuole has an important function in the maintenance of cell volume. Finally, it was observed that the loss of cell viability was not induced by the all-or-none loss of barrier properties.

The asymmetric distribution of chlorpromazine and its quaternary analogue over the erythrocyte membrane

Abstract The binding of chlorpromazine and its quaternary analogue chlorpromazine methoiodide to open and resealed human erythrocyte ghost membranes was studied. The results were compared with binding to liposomes of phosphatidylcholine or phosphatidylcholine with phosphatidylserine. The results indicate that the quaternary compound is confined to the outside face of the membrane. For both compounds two classes of binding sites are available. The strongest binding sites are mainly located on the inner surface of the membrane. The binding data suggest an asymmetric distribution of chlorpromazine in the membrane.

Modulation of neutrophil migration by exogenous gaseous nitric oxide.

We studied the effect of exogenous nitric oxide (NO) on migration of rabbit peritoneal neutrophils. Exogenous NO enhanced random migration of neutrophils in a concentration‐dependent way. An optimally stimulatory effect was observed with 0.5 μM NO, whereas at higher NO concentrations the enhancing effect decreased again. NO caused a rapid and transient increase in intracellular guanosine‐3′,5′‐cyclic monophosphate (cGMP) levels. The enhancing effect of NO on random migration was largely reversed by the inhibitors of cGMP accumulation, LY‐83583 and methylene blue, and by the antagonists of cGMP‐dependent protein kinase, 8‐bromoguanosine‐3′,5′‐cycHc monophosphorothioate, Rp‐isomer (Rp‐8‐Br‐cGMPS) and 8‐(4‐chlorophenylthio)‐guanosine‐3′,5′‐cyclic monophosphorothioate (Rp‐8‐pCPT‐cGMPS). These observations strongly suggest that the enhancement of random migration by NO is mediated by cGMP and cGMP‐dependent protein kinase. The effect of NO on migration did not occur in the absence of extracellular calcium. Although NO did not induce a measurable elevation of intracellular free calcium, preincubation with the intracellular calcium chelator Fura‐2/AM abolished the enhancing effect of NO. It appears therefore that a small change in the level of cytoplasmic free calcium does play a role in the enhancement of random migration by NO. High concentrations of NO were found to inhibit Chemotaxis induced by an optimal concentration of the chemotactic peptide AT‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP). This inhibitory effect was also dependent on the presence of extracellular calcium. A role for cGMP in the inhibition of fMLP‐induced Chemotaxis by NO is not supported by our measurements of intracellular cGMP levels. In contrast to the effects on fMLP, NO did not affect Chemotaxis induced by the phorbol ester PMA. In conclusion, we show that NO, not derived from NO donors but applied directly, may stimulate or inhibit neutrophil migration, dependent on the concentration. The enhancing effect of NO on random migration is mediated by cGMP, which emphasizes the importance of this second messenger as a modulator of neutrophil functions.

The effect of cyclic GMP and cyclic AMP on migration by electroporated human neutrophils

Human neutrophils retain the ability to migrate when they are electroporated; this enables the study of potential mediators by direct application to the cell interior. Cyclic GMP strongly enhances random migration by electroporated human neutrophils. The effect is maximal at a concentration of 10 microM. The potentiating effect of cGMP is chemokinetic. Chemotaxis of electroporated neutrophils activated by formylmethionylleucylphenylalanine (fMLP) is stimulated by cGMP at concentrations up to 10 microM; higher concentrations inhibit chemotaxis. Cyclic AMP resembles cGMP in that both activation and inhibition may occur. However, activation occurs over a very small concentration range, and inhibition is a predominant feature. Cyclic nucleotide-activated migration is dependent on Ca2+, in contrast with activation of migration by fMLP.