Ben M. de Koster

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.

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.

The stimulation of human neutrophil migration by angiotensin II: its dependence on Ca2+ and the involvement of cyclic GMP

Angiotensin II had a bimodal effect on human neutrophil migration. Low concentrations of angiotensin II stimulated random migration. At a concentration of 10−10 M it caused a maximal increase of migration; migration increased from 47.2±2.1 μm in the absence of angiotensin II, to 73.1±2.2 μm with 10−10 M angiotensin II present in the lower compartment of the Boyden chamber (n=5, P<0.001). Stimulation of migration by angiotensin II was partly chemotactic and partly chemokinetic. Angiotensin II concentrations of 10−8 M and higher inhibited chemotactic peptide‐stimulated chemotaxis. The stimulant effect of angiotensin II on migration was completely dependent on extracellular Ca2+. In the presence of 1 mM Ca2+, angiotensin II stimulated migration to 76.1±1.7 μm, while migration in the absence of Ca2+ was 42.2±1.9 μm (n=4, P<0.001). Different types of calcium channel blockers either moderately or strongly inhibited angiotensin II‐activated migration. Stimulation of migration by angiotensin II in intact cells required higher concentrations of Ca2+ than in electroporated cells. This supports the view that there is an influx of Ca2+ through the plasma membrane, and a requirement of calcium for an intracellular target. Angiotensin II‐stimulated migration was inhibited by pertussis toxin; from 71.6±2.0 μm in the absence, to 43.6±1.5 μm in the presence of pertussis toxin (n=4, P<0.001). Migration of electroporated neutrophils stimulated by angiotensin II was synergistically enhanced by GTPγS. This suggests that one or more G‐proteins are involved in the activating effect of angiotensin II. Inhibitors of soluble guanylate cyclase and antagonists of cyclic GMP‐dependent kinase strongly inhibited the activating effect of angiotensin II. The results suggest that the activating effect of angiotensin II is mediated by cyclic GMP and by cyclic GMP‐dependent kinase.

The effect of endothelin-2 (ET-2) on migration and changes in cytosolic free calcium of neutrophils

The effect of endothelin-2 (ET-2) on neutrophil migration and intracellular calcium was studied. Depending on the concentration, ET-2 enhanced or inhibited neutrophil migration. At low concentrations ET-2 caused a chemotactic stimulation of migration, in contrast with endothelin-1 (ET-1) which caused a chemokinetic stimulation of migration. At higher concentrations ET-2 inhibited formyl-methionylleucyl-phenylalanine(fMLP)-activated migration. Both activation and inhibition by ET-2 were completely dependent on extracellular Ca2+. Unlike ET-1 which caused an increase in cytosolic free Ca2+ at a concentration which stimulated migration, ET-2 caused a measurable increase of cytosolic free Ca2+ at a concentration which did not stimulate migration. This strongly suggests that there is no correlation between maximal stimulation of cytoplasmic free calcium, and maximal stimulation of migration. Influx of extracellular Ca2+ was required for both activation of migration and change in cytosolic free Ca+, because no effect was observed in the absence of extracellular Ca+, and because blockers of Ca2+-influx inhibited ET-2-activated migration. The ETA-receptor antagonist cyclo(-D-Trp-D-Asp-Pro-D-Val-Leu) (BQ123), and the ETB-receptor antagonist [Cys11-Cys15]-endothelin-1(11–21) (IRL1038) antagonized the stimulatory effect of ET-2 on migration, and the inhibitory effect of high concentrations of ET-2 on fMLP-activated chemotaxis. This suggests that both the ETA-receptor and the ETB-receptor are involved in the stimulatory effect of low concentrations of ET-2, and in the inhibitory effect of high concentrations of ET-2.

The effect of pentoxifylline on human neutrophil migration: A possible role for cyclic nucleotides

Relatively low concentrations of pentoxifylline caused a stimulation of random migration, while high concentrations inhibited chemotactic migration activated by formyl-methionyl-leucyl-phenylalanine (fMLP). The stimulating effect of pentoxyfylline was partly chemokinetic and partly chemotactic, and was dependent on extracellular calcium. Activation of migration by pentoxifylline was not dependent on the pore size of the micropore filter, indicating that the effect was not mediated by the ability of the drug to induce membrane deformability. Inhibitors of guanylate cyclase and antagonists of cGMP-dependent protein kinase (G-kinase) inhibited stimulation of migration by pentoxifylline. Pentoxyfylline caused a transient increase in cGMP level, while only high concentrations of pentoxifylline caused an increase in cyclic adenosine monophosphate (cAMP) level. It is suggested that the increase of migration is caused by cGMP and is mediated by a G-kinase, while the inhibition of migration at high concentrations of pentoxifylline is mediated by cAMP.

Stimulation of rabbit polymorphonuclear leukocyte locomotion by d-penicillamine

Treatment of polymorphonuclear leukocytes (PMNs) with penicillamine has little effect on chemotaxis of the cells towards fMet-Leu-Phe. In the absence of fMet-Leu-Phe however, penicillamine has a strong effect on PMN locomotion. Penicillamine-induced enhancement of PMN migration is for an important part due to a chemokinetic effect, but there is also a chemotactic effect. Pretreatment of PMNs with membrane-impermeable sulfhydryl reagents has little effect on random locomotion, but completely abolishes the activating effect of penicillamine. This suggests that for the activating effect of penicillamine intact sulfhydryl groups on the outer surface of the PMN are required. Extracellular calcium is no absolute requirement for PMN migration, neither for random locomotion nor for the activating effect of penicillamine. Removal of intracellular Ca2+ with membrane-penetrating Ca2+-complexing agents strongly inhibit penicillamine-induced enhancement of PMN migration, underlining a role for intracellular calcium. Penicillamine causes an increase of cGMP level in PMNs. Though a simple relationship seems unlikely it is possible that the enhancement of PMN migration, and the increase of cGMP level by penicillamine are related processes.

Sodium azide enhances neutrophil migration and exocytosis: involvement of nitric oxide, cyclic GMP and calcium.

Azide, in the absence of other stimuli, enhanced neutrophil migration in a chemotactic way. The effect of azide on migration was significant at concentrations > or = 1 microM and maximal at 10 microM azide. Although azide itself could not induce exocytosis, at concentrations > or = 10 microM azide enhanced exocytosis induced by a combination of the chemotactic peptide f-methionyl-leucyl-phenylalanine (fMLP) and cytochalasin B (CB). Azide can be oxidized by catalase and myeloperoxidase in the presence of H2O2, resulting in the generation of nitric oxide (NO). Formation of NO from azide was detected by ESR spectroscopy with carboxy-PTIO as a NO-selective probe, and by measurement of nitrite formation. Azide-induced migration, and the enhancement by azide of fMLP/CB-induced exocytosis, were blocked by pre-incubating cells with aminotriazole, an inhibitor of catalase and myeloperoxidase, suggesting that the effect of azide was mediated by NO. Azide-induced migration, but not the enhancement by azide of fMLP/CB-induced exocytosis, was inhibited to a large extent by inhibitors of soluble guanylate cyclase and by inhibitors of cGMP-dependent protein kinase. These observations suggest that azide-induced migration is mediated via cGMP and cGMP-dependent protein kinase, while the enhancement of fMLP/CB-induced exocytosis is not. Azide caused a sustained elevation of the intracellular Ca2+-concentration of neutrophils stimulated with fMLP/CB, which was not affected by inhibitors of the cGMP-signalling cascade. Since neutrophil exocytosis has been shown to be closely correlated with increases in intracellular Ca2+, a further increase by azide of the intracellular Ca2+-level of cells stimulated with fMLP/CB provides a likely mechanism for the enhancement of fMLP/CB-induced exocytosis by azide.

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.

The involvement of protein kinase G in stimulation of neutrophil migration by endothelins

Activation of human neutrophil migration by endothelin-1 and endothelin-3 is inhibited by guanylate cyclase inhibitors, by antagonists of protein kinase G (G-kinase), and by KT-5823, an inhibitor of G-kinase. Although no direct effect of endothelins on cGMP level could be established, these results suggest that the effect of these endothelins on migration is mediated by cGMP, and that the effect of cGMP proceeds via a G-kinase. There was little or no effect of guanylate cyclase inhibitors and G-kinase antagonists on endothelin-2-activated migration, indicating that the role of cGMP and G-kinase in endothelin-2-induced activation was either absent or at least different from that of the other endothelins. As compared with other activators, the role of G-kinase in formyl-methionyl-leucyl-phenylalanyl(fMLP-)activated migration resembled that of endothelin-activated migration, while the role of G-kinase in interleukin-8- or leukotriene B4-activated migration was less pronounced.

Ryanodine as inhibitor of chemotactic peptide-induced chemotaxis in human neutrophils

Ryanodine gave a moderate inhibition of chemotactic peptide-activated chemotaxis by intact human neutrophils. Chemotaxis by electroporated neutrophils was strongly inhibited in the nanomolar concentration range. Inhibition of chemotaxis by electroporated neutrophils occurs at concentrations known to open calcium channels in ryanodine-sensitive Ca2+ stores. Whereas migration by formyl-methionyl-leucyl-phenylalanine (fMLP)- or interleukin-8-activated electroporated neutrophils was strongly inhibited by ryanodine, chemotaxis induced by protein kinase C activators was not affected. This suggests that the importance of ryanodine-sensitive Ca2+ stores for migration depends on the type of activator used. Ryanodine gave an increase of cytoplasmic free calcium due to the liberation of calcium from internal stores and to the influx of extracellular calcium. The results show that the neutrophil contains ryanodine-sensitive calcium stores that might be involved in receptor-mediated chemotaxis.