G. J. J. C. Boonen

The role of calcium in neutrophil migration: the effect of calcium and calcium-antagonists in electroporated neutrophils.

Chemotaxis by electroporated rabbit peritoneal neutrophils in the absence of Ca2+ is only slightly different from that in the presence of Ca2+. Pretreatment of neutrophils with quin2-AM causes inhibition of chemotaxis. Calcium antagonists as nitrendipine and verapamil are inhibitory in nanomolar concentrations, while 10(5) times higher concentrations are required for inhibition of chemotaxis by neutrophils which were not electroporated. The results support the hypothesis that Ca2+ from Ca(2+)-storing organelles is of importance for chemotaxis, but that chemotaxis is not dependent on changes in cytoplasmic Ca2+ concentrations.

Neutrophil chemotaxis induced by the diacylglycerol kinase inhibitor R59022

The diacylglycerol kinase inhibitor R59022 induced chemotaxis in neutrophils. The response to R59022 was primarily chemotactic and only very little chemokinetic. Pretreatment with the protein kinase C inhibitors staurosporine and AMG-C16 inhibited chemotaxis induced by R59022 indicating the involvement of protein kinase C. In contrast, chemotaxis induced by fMet-Leu-Phe was only slightly inhibited by staurosporine and AMG16. The effects of R59022 were comparable to the effects of the protein kinase C activators DiC8 and PMA and suggest an involvement of protein kinase C. Pretreatment with pertussis toxin inhibited R59022-induced migration, fMet-Leu-Phe-induced migration, and random migration. GTP gamma S, which stimulates migration of electropermeabilized neutrophils by itself, causes an additive increase of migration in electropermeabilized neutrophils stimulated with a suboptimal concentration R59022, but causes a synergistic increase of migration in cells stimulated with a suboptimal concentration fMet-Leu-Phe. The effects of GTP gamma S on migration are completely inhibited by AMG-C16. This suggests that the GTP-binding protein involved in R59022-activated migration is the G protein that is associated with random migration.

Activation of neutrophil migration by dioctanoyl-sn-glycerol and fMet-Leu-Phe is controlled by different pathways

Migration activated by fMet-Leu-Phe is inhibited by GTP[S] and is little affected by protein kinase C inhibitors. We investigated the effects of GTP[S] and the protein kinase C inhibitor AMG-C16 on dioctanoyl-sn-glycerol (DiC8)-activated migration of rabbit neutrophils and compared them with the effects on fMet-Leu-Phe-activated migration and random migration. GTP[S] did not inhibit DiC8-activated migration or random migration but inhibited fMet-Leu-Phe-activated migration. AMG-C16 gave a strong inhibition of DiC8-activated migration but had only a small effect on fMet-Leu-Phe-activated migration and random migration. When fMet-Leu-Phe and DiC8 were added together in suboptimal concentrations an additive effect was found. Pretreatment with the diacylglycerol kinase inhibitor R59022 enhanced random migration. The enhancement was completely inhibited by AMG-C16 and was unaffected by GTP[S]. These findings suggest that DiC8-activated migration and fMet-Leu-Phe-activated migration are controlled by different pathways.

Inhibition of neutrophil migration by guanine nucleotides.

Guanine nucleotides have, besides an activating effect on exocytosis and respiratory burst in permeabilized neutrophils, a modulating effect on some functions in intact neutrophils. We investigated the effect of guanine nucleotides on fMet-Leu-Phe-induced migration of rabbit neutrophils using the Boyden chamber technique. GTP gave a moderate inhibition of fMet-Leu-Phe-induced neutrophil migration. The GTP analogue GTP[S] had a stronger inhibitory effect than GTP. Other nucleotides, such as GDP, GMP, and guanosine were less effective inhibitors than GTP and GTP[S]. Maximal inhibition was achieved at nucleotide concentration of about 40 μM; higher concentration gave only little additional inhibition. The inhibitory effect persisted when the nucleotide was removed after pretreatment of the neutrophil with that nucleotide. Guanine nucleotide induced inhibition was not due to an interference with the fMet-Leu-Phe receptor as casein-induced migration was equally inhibited. We recently found that ATP inhibited neutrophil chemotaxis. The results obtained with guanine nucleotides resembles the inhibitory effects of ATP and its analogues. It is conceivable that guanine nucleotide-induced inhibition of neutrophil migration is mediated by an interaction of these nucleotides with purinergic receptors.

Cytochalasin B-induced superoxide production in polycation-treated neutrophils

Cytochalasin B alone induces little superoxide production in intact rabbit peritoneal neutrophils. The cytochalasin causes a strong production of superoxide in cells treated with membrane-permeabilizing polycations. Several polycations were able to express the activating effect of cytochalasin B. Especially the poly-l-arginine with a molecular weight of 24,000 proved to be effective. The effectiveness of some polycations is limited because they inactivate the superoxide-generating oxidase system of the neutrophil. Cytochalasin B-induced superoxide production starts at polyl-arginine concentrations that cause a change of membrane permeability. At the concentrations of cytochalasin B used in our experiments, the binding of [3H]cytochalasin B is not enhanced in poly-l-arginine-treated cells as compared with control cells. Activation of superoxide production by cytochalasin B in polycation-treated neutrophils occurs both in the presence or absence of extracellular Ca2+. When the cells are pretreated with agents that known to interfere with intracellular Ca2+, the subsequent activation is strongly inhibited, suggesting a role for intracellular Ca2+ in cytochalasin B-induced activation. It is suggested that cytochalasin B alone is not able to activate all the steps that eventually result in complete activation of the superoxide-generating oxidase and that membrane perturbation by polycation provides activation of the remaining steps.

Induction of superoxide production in rabbit neutrophils by a stable analogue of GTP

Treatment of rabbit neutrophils with the stable GTP analogue GTPγS results in generation of superoxide. Whereas poly-l-arginine, an agent known to damage the plasma membrane, has only a small effect by itself, a strong and synergistic enhancement of superoxide generation is obtained in the presence of GTPγS and poly-l-arginine. The effect is potentiated in the presence of NADPH. Other nucleotides, such as GTP, are not effective in inducing superoxide generation. The results indicate that the stable guanine nucleotide GTPγS may activate superoxide production in rabbit neutrophils, and that this effect is evident if the plasma membrane has been permeabilized by poly-l-arginine, allowing an easy entry of GTPγS into the cell.