Inhibition of neutrophil migration soon after initiation of treatment with leflunomide or methotrexate in patients with rheumatoid arthritis: findings in a prospective, randomized, double-blind clinical trial in fifteen patients.

OBJECTIVE: Leflunomide is a novel immunomodulating drug that has recently been approved as a disease-modifying antirheumatic drug for the treatment of rheumatoid arthritis (RA). The aim of this study was to determine the relationship between the clinical effects of leflunomide and neutrophil migration. METHODS: The effects of leflunomide and methotrexate on neutrophil chemotaxis were studied in 15 RA patients who participated in a prospective, randomized, double-blind clinical trial. When possible, neutrophil numbers were counted in synovial fluid (SF) samples at baseline and after 14 days, 4 months, and 1 year of treatment. The chemotactic properties of peripheral blood neutrophils from RA patients treated with either leflunomide or methotrexate were studied by the Boyden chamber technique, using the activators formyl-methionyl-leucyl-phenylalanine (fMLP) and interleukin-8 (IL-8). The in vitro effects of A77 1726, the active metabolite of leflunomide, and methotrexate on peripheral blood neutrophils from 7 healthy control subjects were also investigated. RESULTS: Both therapy groups exhibited clinical improvement, including rapid reductions in SF neutrophil counts and reduced joint swelling and tenderness. On day 14, 3 of 7 patients who received leflunomide showed no detectable effusions. There was a significant effect on neutrophil chemotaxis (P < 0.001), which was similar for leflunomide and methotrexate. The direct effects on the neutrophils diminished over time. Incubation of peripheral blood neutrophils from healthy controls with A77 1726 confirmed the inhibitory effect on chemotaxis. CONCLUSION: Leflunomide treatment is beneficial in RA patients. Different mechanisms are operative in various phases of treatment, leading to decreased recruitment of inflammatory cells in the joints.

Inhibition of interleukin-8-activated human neutrophil chemotaxis by thapsigargin in a calcium- and cyclic AMP-dependent way.

Chemotactic migration of human neutrophils, induced by interleukin-8 (IL-8) or other activators, was inhibited by thapsigargin in the high nanomolar range. The degree of inhibition depended on the type of activator. Other inhibitors of Ca(2+)-ATPases associated with intracellular calcium stores, such as cyclopiazonic acid and 2,5-di-(tert-butyl)-1,4-benzohydroquinone, equally inhibited IL-8-activated migration. Inhibition of migration by thapsigargin and the other ATPase inhibitors occurred only in the presence of extracellular Ca2+; migration was not inhibited in the presence of EGTA. La3+ reversed thapsigargin-induced inhibition to a large degree; other calcium channel blockers gave a partial reversal (econazole, verapamil, and SK&F 96365) or had no effect (gadolinium chloride and Ni2+). Using electroporated cells and Ca buffers, it was shown that inhibition started at about 0.2 microM and was complete at a cytosolic Ca concentration of about 2 microM. It appears that under certain conditions the thapsigargin-induced influx of extracellular calcium, causing relatively high local calcium concentrations, initiates or permits a process which may be detrimental to chemotactic migration. Cyclic AMP (cAMP; adenosine 3',5'-cyclic monophosphate) is probably involved in this process, because thapsigargin increased the cAMP level and cAMP inhibited IL-8-activated migration in a calcium-dependent way. The hypothesis that cAMP is involved in the effect of thapsigargin on migration is supported by the finding that very low concentrations of thapsigargin stimulate neutrophil migration in the absence of other chemoattractants. The results suggest that thapsigargin causes a (compartmentalized) increase in cAMP, which results in a calcium-dependent modulation of migration.

The effects of photodynamic therapy on human neutrophil migration using bacteriochlorin a.

Bacteriochlorin a photodynamic therapy (BCA-PDT) caused inhibition of interleukin (IL)-8-activated neutrophil migration, at concentrations that did not induce membrane damage. Random migration and migration induced by other chemoattractants were also inhibited, indicating that the effect of BCA-PDT was not at the level of chemoattractant-receptor interaction but down stream. The BCA-PDT completely blocked superoxide production of phorbol ester-stimulated neutrophils indicating that superoxide production by neutrophils present in the tumor before and during BCA-PDT is not the cause of inactivation of tumor cells. Both type I and type II quenchers prevented inhibition by BCA-PDT but only in electroporated cells. Confocal laser scanning microscopy showed that the fluorescence of BCA was located inside the cell. These results show that the effects of BCA-PDT are intracellular and of a mixed type I/type II character and that the neutrophils present in the tumor during illumination probably do not contribute to tumor eradication by releasing reactive oxygen species.

The role of cyclic AMP and protein kinase A in stimulation of neutrophil migration by endothelins.

The role of cAMP and cAMP-dependent protein kinase A (PKA) in endothelin (ET)-stimulated migration of human neutrophils was studied. Endothelins caused an increase in neutrophil migration when they were applied in low (nanomolar) concentrations; stimulation of migration was either predominantly chemokinetic (ET-1) or chemotactic (ET-2, ET-3). All endothelins, at concentrations which gave maximal stimulation of migration, caused an increase of cAMP level. Two inhibitors of adenylate cyclase, MDL-12330A and SQ-22536, completely inhibited migration activated by ET-1, ET-2 or ET-3, indicating that cAMP generation played a decisive role in endothelin-activated migration. The role of PKA in endothelin-activated migration was considered. Two specific antagonists of PKA strongly inhibited endothelin-activated migration. KT-5720, an inhibitor of PKA, also inhibited ET-activated migration, but only when electroporated cells were used. The results suggest that the effect of cAMP on endothelin-activated migration was mediated by PKA.

Interaction of cyclic GMP and cyclic AMP during neutrophil migration: involvement of phosphodiesterase type III.

In previous experiments, it was shown that migration of electropermeabilized human neutrophils induced by a combination of cGMP and cAMP markedly lower relative to that induced by cGMP or cAMP alone. However, when cGMP was replaced with 8-(para-chlorophenylthio-guanosine-3',5'-cyclic monophosphate (8-pCPT-cGMP), a metabolic stable analogue of cGMP which does not affect the activity of cGMP-regulated phosphodiesterases (PDEs), migration in the presence of cAMP was enhanced in an additive way. To investigate the role of cyclic nucleotide breakdown during neutrophil migration in more detail, specific inhibitors of phosphodiesterase type III (PDE-III) (cGMP-inhibited) were used. Milrinone and cilostamide inhibited migration induced by an optimal concentration of cAMP. This revealed that inhibition of cAMP breakdown, by prolonging the action of an otherwise optimal concentration of cAMP, led to decreased migration, in accordance with the observation that the effect of cAMP on migration of electropermeabilized neutrophils was biphasic. Furthermore, it was found that a combination of 8-pCPT-cGMP and milrinone/cilostamide could substitute for cGMP in both activating cGMP-dependent protein kinase (8-pCPT-cGMP) and inhibiting PDE-III (milrinone/cilostamide). In conclusion, evidence is presented that cGMP and cAMP could interact on the level of PDE-III during neutrophil 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.

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.

Intracellular but not extracellular conversion of nitroxyl anion into nitric oxide leads to stimulation of human neutrophil migration.

Considerable controversy exists in the literature with regard to the nature of the agent mediating the biological effects of nitroxyl (NO-) donors. Here it is demonstrated that Angeli's salt (AS), a generator of NO-, enhanced human neutrophil migration. Under aerobic conditions, AS was converted to peroxynitrite to a small extent. However, using methionine, a scavenger of peroxynitrite, it was shown that peroxynitrite was not involved in AS-induced migration. AS equally enhanced human neutrophil migration under aerobic and anaerobic conditions, which strongly suggests that extracellular conversion of NO- to .NO by oxygen was not required. Furthermore, metHb and L-cysteine, which react more readily with NO- than with .NO, inhibited AS-induced migration, whereas the response towards gaseous .NO remained unaffected. AS induced an increase in the intracellular level of cGMP, although the curves for migration and cGMP level appeared to be slightly different in their concentration dependence. An inhibitor of soluble guanylate cyclase and antagonists of cGMP-dependent protein kinase had a more pronounced inhibitory effect on .NO-induced migration than on AS-induced migration. This suggests that the cGMP signalling cascade is partially, but not solely, responsible for AS-induced migration. As it has been demonstrated that soluble guanylate cyclase can only be activated by .NO, and not by NO-, these data indicate that NO- is at least partly converted intracellularly to .NO.

The effect of thimerosal on neutrophil migration: a comparison with the effect on calcium mobilization and CD11b expression.

The sulfhydryl-reactive compound thimerosal caused a chemotactic stimulation of neutrophil migration at low concentrations and inhibition of chemoattractant-stimulated chemotaxis at high concentrations. Thiosalicylic acid, an analog of thimerosal devoid of mercury, also stimulated migration at low concentrations and caused inhibition at higher concentrations, though the inhibitory effect was less pronounced than that of thimerosal. These results indicate that the stimulatory effect of thimerosal on migration is due to the thiosalicylic acid moiety of the molecule. In contrast with thimerosal which, especially at higher concentrations than required for optimal stimulation of migration, caused an increase in cytosolic free calcium ([Ca2+]i), thiosalicylic acid had no effect on [Ca2+]i of the neutrophil. This suggests that the presence of mercury is decisive for the calcium-mobilizing effect, but not for stimulation of migration, and that mobilization of calcium and activation of migration are not related. Thimerosal caused a strong increase of CD11b expression in neutrophils in suspension, especially at inhibitory concentrations, while thiosalicylic acid had no effect on CD11b expression. This could mean (but does not prove) that CD11b expression is more related to the calcium-mobilizing effect of thimerosal than to its stimulation of migration.

N-acetylcysteine causes a transient stimulation of neutrophil migration.

Random migration of rabbit peritoneal neutrophils was enhanced in a chemokinetic way by N-acetylcysteine (NAC) in a small concentration range (10-400 microM). The enhancement was due to the cysteine moiety in the molecule, because cysteine equally caused a stimulation of random migration. The stimulating effect of NAC or cysteine largely disappeared when cells were preincubated with NAC or cysteine for 30 min before submission to chemotaxis, indicating that desensitization occurs. The stimulating effect of NAC was dependent on extracellular calcium. Because the Ca2+-dependence of migration by electroporated cells differed from that of intact cells, and because calcium channel blockers inhibited the effect of NAC, the calcium-dependent target is probably located inside the cell rather than on the cell surface. In contrast with fMLP, NAC did not cause an upregulation of CD11b expression of cells in suspension. Inhibitors of guanylate cyclase and of cGMP-dependent protein kinase (G-kinase) inhibited stimulation of migration by NAC, suggesting that cGMP played a decisive role in the stimulatory effect of NAC.

Cyclic nucleotide-induced migration of electropermeabilized neutrophils: the effect of phosphodiesterase-resistant analogues.

OBJECTIVE: To study the effect of cyclic nucleotides and PDE-resistant cyclic nucleotide analogues on neutrophil migration. METHODS: Migration of electropermeabilized neutrophils in Boyden chambers in vitro. RESULTS: Addition of cyclic AMP inhibited migration of electropermeabilized neutrophils in the presence of cGMP, relative to the level of migration in the presence of cGMP alone. However, when cGMP was replaced with 8-pCPT-cGMP or Sp-cGMPS, analogues of cGMP which are not degraded by phosphodiesterases, cAMP synergistically enhanced migration. In contrast, migration in the presence of the phosphodiesterase-resistant cAMP analogue, Sp-cAMPS, was not enhanced by addition of cGMP. CONCLUSIONS: Taking into account reports in the literature which show that cGMP-hydrolysing activity can be enhanced by the catalytic subunit of cAMP-dependent protein kinase, it is hypothesized that breakdown of cGMP in neutrophils may be modulated via cAMP.

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.

A cyclic GMP- and G-kinase-dependent effect of azathioprine on migration by human neutrophils.

Relatively high concentrations of azathioprine had an inhibitory effect on interleukin 8 (IL-8)- or formyl-methionyl-leucyl-phenylalanine-activated (fMLP)-chemotaxis by human neutrophils. However, application of low concentrations of azathioprine in a concentration gradient gave a chemotactic stimulation to random migration. Stimulation of migration was maximal at a concentration of 5 microM azathioprine; at higher concentrations stimulation decreased again. The activating effect of azathioprine is located in the mercaptopurine moiety of the molecule, since mercaptopurine also stimulated neutrophil migration. In contrast to some other chemotactic agents such as fMLP and IL-8, an activating concentration (5 microM) of azathioprine did not cause an upregulation of CD11b expression on neutrophils in suspension. High concentrations of azathioprine (1 mM) inhibited CD11b expression of fMLP- or IL-8- activated neutrophils; the latter could explain the inhibitory effect of azathioprine. Azathioprine caused a transient stimulation of cGMP level; inhibitors of guanylate cyclase inhibited azathioprine-stimulated migration, suggesting that cGMP was associated with the stimulating effect of azathioprine on migration. Antagonists of cGMP-dependent protein kinase (G-kinase) strongly inhibited azathioprine-activated migration, indicating that the effect of azathioprine proceeds via G-kinase. The antagonists had only a marginal effect on inhibition of IL-8-activated chemotaxis by high concentrations of azathioprine, thus the G-kinase seems not to be of great importance on the inhibitory effect of azathioprine.

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

1. 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 microns in the absence of angiotensin II, to 73.1 +/- 2.2 microns 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. 2. 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 microns, while migration in the absence of Ca2+ was 42.2 +/- 1.9 microns (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. 3. Angiotensin II-stimulated migration was inhibited by pertussis toxin; from 71.6 +/- 2.0 microns in the absence, to 43.6 +/- 1.5 microns in the presence of pertussis toxin (n = 4, P < 0.001). Migration of electroporated neutrophils stimulated by angiotensin II was synergistically enhanced by GTP gamma S. This suggests that one or more G-proteins are involved in the activating effect of angiotensin II. 4. 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 sodium aurothiomalate on stimulated and non-stimulated migration by human neutrophils: the role of cyclic GMP.

Preincubation of human neutrophils with aurothiomalate had little effect on random migration or chemotactic migration towards the chemotactic peptide fMLP. However, a strong enhancement of migration was observed when aurothiomalate was present in a concentration gradient; the effect of the drug was chemotactic rather than chemokinetic. Thiomalate also caused a chemotactic enhancement of migration but here a tenfold higher concentration of the drug was required as compared with aurothiomalate. Aurothiomalate caused an increase of cellular cGMP level, and inhibitors of guanylate cyclase inhibited the activating effect of aurothiomalate. Three specific antagonists of cGMP-dependent kinase (G-kinase) strongly inhibited aurothiomalate-induced migration of electroporated neutrophils. The results suggest that stimulation of migration by aurothiomalate is mediated by cGMP and a G-kinase. Monoclonal anti-IL-8 inhibited aurothiomalate-induced stimulation of migration. Though no increased release of IL-8 could be established upon exposure of neutrophils to aurothiomalate, it seems conceivable that the stimulating effect of aurothiomalate is mediated by IL-8.

Carbon monoxide enhances human neutrophil migration in a cyclic GMP-dependent way.

Carbon monoxide (CO) enhanced random migration of human neutrophils. An optimally stimulatory effect was observed with 10 microM CO. CO caused a rapid and transient increase in intracellular level of guanosine-3',5'-cyclic monophosphate (cGMP). The enhancing effect of CO on random migration was reversed to a large extent by inhibitors of cGMP accumulation, and by antagonists of cGMP-dependent protein kinase (G-kinase). These results strongly suggest that the enhancement of random migration by CO is mediated by cGMP and G-kinase. Using hemoglobin, a scavenger of CO, we could show that stimulation of soluble guanylate cyclase over an extended period of time, rather than the observed fast and transient increase in intracellular cGMP levels, is responsible for CO-activated migration. We postulate that CO, like nitric oxide (NO), acts as a biological signal in the immune system.

Modulation of human neutrophil chemotaxis by the endothelin-B receptor agonist sarafotoxin S6c.

At low concentrations (4 x 10(-9) M) sarafotoxin S6c, a selective agonist of the ETB receptor, caused a slight but significant chemotactic enhancement of neutrophil migration. In contrast with stimulation by endothelins, stimulation of migration by sarafotoxin was only mediated by the ETB receptor because BQ123, a selective antagonist of the ETA receptor, had no effect on the enhancement. At higher concentrations (10(-8) M and higher) sarafotoxin inhibited neutrophil migration stimulated by chemotactic activators. It thus appears that the ETB receptor mediates the inhibition of migration by high concentrations of sarafotoxin. The degree of inhibition by sarafotoxin was dependent on the type of activator; especially IL-8 activated migration was strongly inhibited. Extracellular calcium had little effect on stimulation or inhibition by sarafotoxin, in contrast with endothelins. In a very small concentration range (4 x 10(-8) M) sarafotoxin caused an increase of cytoplasmic free calcium; higher concentrations of sarafotoxin had no effect on cytoplasmic calcium, nor did they affect calcium changes caused by fMet-Leu-Phe (fMLP). IRL1038, a selective antagonist of the ETB receptor, equally gave a small potentiation at low concentrations, and inhibited agonist-activated chemotaxis at higher concentrations, suggesting that occupation of the ETB receptor by either agonist or antagonist results in a modulation of migration.

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 microM 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'-cyclic 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, pre-incubation 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 N-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 functional.

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-methionyl-leucyl-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 Ca2+, because no effect was observed in the absence of extracellular Ca2+, and because blockers of Ca(2+)-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.

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.