FK-506, a potent novel inhibitor of the release of proinflammatory mediators from human Fc epsilon RI+ cells.

FK-506, a macrolide that binds with high affinity to a specific binding protein, and the structurally related macrolide rapamycin (RAP) were compared to cyclosporin A (CsA) for their effects on the release of preformed (histamine) and de novo synthesized (peptide leukotriene C4) inflammatory mediators from human basophils. FK-506 (1 to 300 nM) concentration dependently inhibited histamine release from basophils activated by Der p I Ag, anti-IgE, or compound A23187. FK-506 was more potent than CsA when basophils were challenged with Ag (IC50 = 25.5 +/- 9.5 vs 834.3 +/- 79.8 nM; p less than 0.001), anti-IgE (IC50 = 9.4 +/- 1.7 vs 441.3 +/- 106.7 nM; p less than 0.001), and A23187 (IC50 = 4.1 +/- 0.9 vs 36.7 +/- 3.8 nM; p less than 0.001). The maximal inhibitory effect of FK-506 was higher than that caused by CsA when basophils were activated by Der p I (80.0 +/- 3.6 vs 49.5 +/- 4.7%; p less than 0.001) and anti-IgE (90.4 +/- 1.8 vs 62.3 +/- 2.9%; p less than 0.001). FK-506 had little or no effect on the release of histamine caused by f-met peptide, phorbol myristate (12-tetradecanoyloxy-13-acetoxy-phorbol), and bryostatin 1. RAP (30 to 1000 nM) selectively inhibited only IgE-mediated histamine release from basophils, although it had no effect on mediator release caused by f-met peptide, A23187, 12-tetradecanoyloxy-13-acetoxy-phorbol, and bryostatin 1. FK-506 also inhibited the de novo synthesis of sulfidopeptide leukotriene C4 from basophils challenged with anti-IgE. Low concentrations of FK-506 and CsA synergistically inhibited the release of mediators from basophils induced by anti-IgE or compound A23187. IL-3 (3 and 10 ng/ml), but not IL-1 beta (10 and 100 ng/ml), reversed the inhibitory effect of both FK-506 and CsA on basophils challenged with anti-IgE or A23187. RAP was a competitive antagonist of the inhibitory effect of FK-506 on A23187-induced histamine release from basophils with a dissociation constant of about 30 nM. In contrast, RAP did not modify the inhibitory effect of CsA on A23187-induced histamine release. These data indicate that FK-506 is a potent antiinflammatory agent that acts on human basophils presumably by binding to a receptor site (i.e., FK-506 binding protein).     

The human recombinant c-kit receptor ligand, rhSCF, induces mediator release from human cutaneous mast cells and enhances IgE-dependent mediator release from both skin mast cells and peripheral blood basophils.

The gene product of the steel locus of the mouse represents a growth factor for murine mast cells and a ligand for the c-kit proto-oncogene receptor, a member of the tyrosine kinase receptor class of oncogenes (for review, see O. N. Witte. 1990. Cell 63:5). We have studied the effect of the human recombinant c-kit receptor ligand stem cell factor (rhSCF) on the release of inflammatory mediators from human skin mast cells and peripheral blood basophils and compared its activity to that of rhIL-3, rhSCF (1 ng/ml to 1 microgram/ml) activated the release of histamine and PGD2 from mast cells isolated from human skin. Analysis by digital video microscopy indicated that purified human skin mast cells (84 +/- 5% pure) responded to rhSCF (0.1 to 1 microgram/ml) challenge with a rapid, sustained rise in intracellular Ca2+ levels that was accompanied by secretion of histamine. A brief preincubation (10 min) of mast cells with rhSCF (0.1 pg/ml to 1 ng/ml) significantly enhanced (100 +/- 35%) the release of histamine induced by anti-IgE (3 micrograms/ml), but was much less effective on IgE-mediated release of PGD2. In contrast, a short term incubation with rhSCF did not potentiate the secretion of histamine activated by substance P (5 microM). A 24-h incubation of mast cells with rhSCF did not affect the release of mediators induced by anti-IgE (3 micrograms/ml), probably due to receptor desensitization, rhSCF (1 ng/ml to 3 micrograms/ml) neither caused release of histamine or leukotriene C4 (LTC4) release from leukocytes of 14 donors, nor induced a rise in intracellular Ca2+ levels in purified (greater than 70%) basophils. Brief preincubation (10 min) of leukocytes with rhSCF (1 ng/ml to 3 micrograms/ml) caused an enhancement (69 +/- 11%) of anti-IgE-induced release of histamine that was significant at concentrations as low as 3 ng/ml (p less than 0.05), whereas it appeared less effective in potentiating IgE-mediated LTC4 release. In contrast, a prolonged incubation (24 h) with rhSCF (0.1 pg/ml to 100 ng/ml) did not enhance the release of histamine or LTC4 induced by anti-IgE (0.1 microgram/ml), whereas rhIL-3 (3 ng/ml) significantly potentiated the release of both mediators.(ABSTRACT TRUNCATED AT 400 WORDS)     

Human basophil releasability. III. Genetic control of human basophil releasability

Basophil releasability implies that, in addition to the surface density of IgE molecules, biochemical events determine the capacity to release chemical mediators in response to activating stimuli. We studied the IgE (anti-IgE)-mediated and non-IgE-mediated (f-met peptide and the Ca2+ ionophore A23187) releasability of human basophils obtained from 14 monozygotic (MZ) (ages 25.7 +/- 13.3 yr; mean +/- SDM) and 13 dizygotic (DZ) twin pairs (ages 20.4 +/- 9.9 yr). A significant intrapair correlation coefficient of the maximal percent of anti-IgE-induced histamine release was found in the MZ, whereas no significant correlation was found in the DZ. The mean intrapair variance of anti-IgE-induced histamine release in MZ (VMZ) and in DZ (VDZ) gave an F value equal to 3.84 (p less than 0.01) and a heritability (H) index of 0.74. Similar findings were obtained with respect to the sensitivity to a standard concentration (10(-1) micrograms/ml) of anti-IgE. No correlation between serum IgE level and anti-IgE-induced histamine release was found in either MZ or DZ. A significant intrapair correlation coefficient of f-met peptide-induced histamine release was found in both the MZ and the DZ. The difference between MZ and DZ was not significant. The VMZ and the VDZ of the f-met peptide-induced histamine release gave an F value of 1.52 (NS) and an H value of 0.34. The intrapair correlation coefficient of A23187-induced release was significant in MZ and not significant in DZ. The mean intrapair variance of A23187-induced histamine release gave an F value of 2.33 (NS) and an H index of 0.57. Similar findings were obtained by using suboptimal (3 X 10(-1) micrograms/ml) concentrations of A23187. There was no correlation between the sensitivity of basophils to release in response to anti-IgE and their response to f-met peptide or A23187, in either the MZ or the DZ. We conclude that the ability of basophils to respond to anti-IgE and A23187 is influenced by genetic factors.     

Cyclosporin A rapidly inhibits mediator release from human basophils presumably by interacting with cyclophilin.

We have examined the effects of cyclosporin A (CsA) and a series of CsA analogs that bind with decreasing affinity to cyclophilin, to evaluate the involvement of this protein in the release of preformed (histamine) and de novo synthesized (peptide leukotriene C4; LTC4) mediators of inflammatory reactions from human basophils. CsA (8 to 800 nM) concentration-dependently inhibited (5 to 60%) histamine release from peripheral blood basophils challenged with anti-IgE. CsA was more potent (92.6 +/- 1.8 vs 59.1 +/- 4.5%; p less than 0.001) and, at low concentrations, more effective when the channel-operated influx of Ca2+ was bypassed by the ionophore A23187 (IC40 = 24.1 +/- 3.9 vs 105.5 +/- 22.2 nM; p less than 0.05). CsA had no effect on the release of histamine caused by phorbol myristate and bryostatin 1 that activate different isoforms of protein kinase C. Inhibition of histamine release from basophils challenged with anti-IgE was not abolished by washing (three times) the cells before anti-IgE challenge. CsA also inhibited the de novo synthesis of LTC4 from basophils challenged with anti-IgE. The inhibitory effect of CsA was very rapid, and the drug, added from 1 to 10 min during the reaction, inhibited the ongoing release of histamine caused by anti-IgE and by A23187. The experiments with CsA analogs (CsG, CsC, CsD, and CsH) showed that CsH, which has an extremely low affinity for cyclophilin, has no effect on basophil mediator release. In addition, there is a significant correlation between the concentrations of CsA, G, C, and D that inhibited by 30% the histamine release induced by anti-IgE (r = 0.99; p less than 0.001) and by A23187 (r = 0.87; p less than 0.001) and their affinity for cyclophilin.     

Protein kinase C (PKC) changes in human basophils. IgE-mediated activation is accompanied by an increase in total PKC activity

We have examined the changes in protein kinase C (PKC) which follow IgE-mediated activation of basophils. Exposure to 0.1 microgram/ml anti-IgE resulted in an increase in total cellular PKC (169 +/- 23% of control, histamine release (HR) = 33 +/- 7%, n = 12) which could be accounted for solely by the increase in membrane-associated PKC. These changes reached a maximum (280 +/- 48%) 1.0 min after challenge and declined to 190 +/- 38% after 5.0 min though histamine release was not complete until 5 to 10 min later. We found a good correlation between the increase in membrane-associated PKC and the eventual release of histamine (rs = 0.902). Donors whose basophils released less than 5% total histamine (n = 3, HR = 3 +/- 1%) showed a partial activation of PKC (173 +/- 18%) though much less than the remaining donors (increase in PKC = 346 +/- 59%, n = 9, HR = 43 +/- 7%). We observed no redistribution of cytosolic PKC at any time following exposure to anti-IgE. In contrast, 0.1 microgram/ml 2-O-tetradecanoyl-phorbol-13-acetate (HR = 36 +/- 3%, n = 3) promoted an increase in total cellular PKC, the loss of 31 +/- 4% of the cytosolic PKC and an 816 +/- 183% increase in membrane-associated PKC. Activation of PKC by anti-IgE was only partially dependent on extracellular calcium. In the absence of calcium, the increase in PKC was approximately 65% (n = 4) of that noted in the presence of 1mM calcium but these levels were sustained over much longer periods, failing to return to base line after 30 min. Higher than normal concentrations of calcium (5 to 10 mM) promoted rapid increases in PKC activity and accelerated the return to base line (back to prechallenge levels by 5 min). Suboptimal concentrations of anti-IgE (0.01 microgram/ml) attenuated the changes in membrane associated PKC and altered the kinetics of the response. The time required to reach maximum activity increased from 1.0 to 5.0 min with a corresponding decrease in the rate at which histamine was released. Higher concentrations of anti-IgE (1.0 microgram/ml) promoted a rapid increase in PKC (maximum increase in PKC = 501 +/- 59%, time = 0.5 min, HR = 28 +/- 2%) followed by an equally rapid return to base line levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolic comparison between basophils and other leukocytes from human blood

Basophilic granulocytes were purified from the blood of normal individuals by successive isopyknic centrifugation and elutriation centrifugation. Starting with the leukocyte-rich fraction of 500 ml of blood, we recovered 31 to 80% (mean 51%, n = 20) of the basophils in 45 to 87% purity (mean 69%, n = 23). The contaminating cells were mainly lymphocytes. The basophils were greater than 98% vital (exclusion of ethidium bromide and hydrolysis of fluorescein diacetate). The histamine content of the basophils was 1.1 to 2 pg/cell (mean 1.6 pg/cell, n = 22). With anti-IgE, 30 to 50% of the histamine was released; with phorbol myristic acetate (PMA) or the calcium ionophore A23187, 70 to 100% of the histamine was released. Serum-opsonized zymosan (STZ) did not induce histamine release. Reactions with monoclonal antibodies revealed that the basophils expressed the C3bi receptor (CR3) and the leukocyte function-associated antigen 1 (LFA1), but not the gp 150,95 antigen, the C3b receptor (CR1), or the low avidity Fc gamma receptor. Basophils carry class I but not class II HLA antigens. During incubation of the basophils with serum-opsonized Staphylococcus aureus or Escherichia coli, these bacteria were neither phagocytized nor killed. STZ, PMA, A23187, or anti-IgE did not initiate an "oxidative burst" in the basophils. This was tested with oxygen consumption, cytochrome c reduction, NBT reduction, chemiluminescence, and release of hydrogen peroxide. Moreover, we did not detect cytochrome b558, superoxide dismutase, catalase, or peroxidase in the basophils. Of the typical granule-associated enzymes lysozyme, Vitamin B12-binding protein, and beta-glucuronidase, only beta-glucuronidase was present in the basophils in detectable amounts. This enzyme was released, together with histamine, on incubation of the cells with PMA, A23187, or anti-IgE, but not with STZ. We conclude that basophils from normal human blood are not phagocytes and are probably not involved in the oxidative defense of the host against foreign antigens.     

Effects of ADP-ribosylation of GTP-binding protein by pertussis toxin on immunoglobulin E-dependent and -independent histamine release from mast cells and basophils

Pretreatment of rat peritoneal mast cells, human basophils, bone marrow-derived mouse mast cells (BMMC) and mouse mast cell line PT-18 cells with 1 microgram/ml pertussis toxin (PT) failed to inhibit immunoglobulin E (IgE)-dependent histamine release from the cells. In BMMC and PT-18 cells, even 20-hr incubation of the cells with 1 microgram/ml PT, which ADP-ribosylates more than 97% of 41 kDa, alpha-subunit of Ni in the cells, failed to affect the IgE-dependent release of histamine or arachidonate. The results indicate that GTP-binding protein, Ni, is not involved in the transduction of triggering signals induced by cross-linking of IgE receptors. In contrast, pretreatment of rat mast cells with 1 ng/ml to 0.1 microgram/ml PT for 2 hr inhibited histamine release induced by compound 48/80 in a dose-dependent manner. A similar pretreatment with PT inhibited thrombin-induced histamine release from BMMC and N-formyl-L-methionyl-L-leucyl-L-phenylalanine-induced histamine release from human basophils in a similar dose-dependent fashion. However, even 20 hr of incubation of sensitized BMMC with 1 microgram/ml PT failed to inhibit either thrombin-induced or antigen-induced breakdown of phosphatidylinositides (PI), i.e., the formation of inositol triphosphate and diacylglycerol, Quin-2 signal, and the release of arachidonic acid. The results indicate that the inhibition of thrombin-induced histamine release by PT-treatment is not due to the inhibition of PI-turnover, and that Ni is not involved in thrombin-induced or antigen-induced (IgE-dependent) hydrolysis of phosphatidylinositides in mast cells.     

Protein L. A bacterial Ig-binding protein that activates human basophils and mast cells.

Peptostreptococcus magnus strain 312 (10(6) to 10(8)/ml), which synthesizes a protein capable of binding to kappa L chains of human Ig (protein L), stimulated the release of histamine from human basophils in vitro. P. magnus strain 644, which does not synthesize protein L, did not induce histamine secretion. Soluble protein L (3 x 10(-2) to 3 micrograms/ml) induced histamine release from human basophils. The characteristics of the release reaction were similar to those of rabbit IgG anti-Fc fragment of human IgE (anti-IgE): it was Ca2(+)- and temperature-dependent, optimal release occurring at 37 degrees C in the presence of 1.0 mM extracellular Ca2+. There was an excellent correlation (r = 0.82; p less than 0.001) between the maximal percent histamine release induced by protein L and that induced by anti-IgE, as well as between protein L and protein A from Staphylococcus aureus (r = 0.52; p less than 0.01). Preincubation of basophils with either protein L or anti-IgE resulted in complete cross-desensitization to a subsequent challenge with the heterologous stimulus. IgE purified from myeloma patients PS and PP (lambda-chains) blocked anti-IgE-induced histamine release but failed to block the histamine releasing activity of protein L. In contrast, IgE purified from myeloma patient ADZ (kappa-chains) blocked both anti-IgE- and protein L-induced releases, whereas human polyclonal IgG selectively blocked protein L-induced secretion. Protein L acted as a complete secretagogue, i.e., it activated basophils to release sulfidopeptide leukotriene C4 as well as histamine. Protein L (10(-1) to 3 micrograms/ml) also induced the release of preformed (histamine) and de novo synthesized mediators (leukotriene C4 and/or PGD2) from mast cells isolated from lung parenchyma and skin tissues. Intradermal injections of protein L (0.01 to 10 micrograms/ml) in nonallergic subjects caused a dose-dependent wheal-and-flare reaction. Protein L activates human basophils and mast cells in vitro and in vivo presumably by interacting with kappa L chains of the IgE isotype.     

Characterization of the anti-inflammatory effect of FK-506 on human mast cells.

We have examined the effects of FK-506 and of the struturally related macrolide rapamycin, which bind with high affinity to a specific binding protein (FKBP), to evaluate the involvement of this protein in the release of preformed (histamine) and de novo synthesized inflammatory mediators (sulfidopeptide leukotriene C4 and prostaglandin D2) from mast cells isolated from human lung parenchyma. FK-506 (0.1 to 300 nM) concentration dependently inhibited histamine release from lung parenchymal mast cells activated by anti-IgE. FK-506 was more potent in lung mast cells than in basophils (IC50 = 1.13 +/- 0.46 nM vs 5.28 +/- 0.88 nM; p less than 0.001), whereas the maximal inhibitory effect was higher in basophils than in lung mast cells (88.4 +/- 2.5% vs 76.4 +/- 3.8%; p less than 0.01). FK-506 had little or no inhibitory effect on histamine release from lung mast cells challenged with compound A23187, whereas it completely suppressed A23187-induced histamine release from basophils. FK-506 also inhibited the de novo synthesis of 5-lipoxygenase (sulfidopeptide leukotriene C4) and cyclo-oxygenase (prostaglandin D2) metabolites of arachidonic acid from mast cells challenged with anti-IgE. Unlike in basophils, Il-3 (3 to 30 ng/ml) did not modify anti-IgE- or A23187-induced histamine release from lung mast cells nor did it reverse the inhibitory effect of FK-506. Rapamycin (3 to 300 nM) had little or no effect on the release of histamine from lung mast cells, but it was a competitive antagonist of the inhibitory effect of FK-506 on anti-IgE-induced histamine release from human mast cells with a dissociation constant of about 12 nM. These data indicate that FK-506 is a potent anti-inflammatory agent that acts on human lung mast cells presumably by binding to a receptor site (i.e., FKBP).     

Leukotriene C4 formation by enriched human basophil preparations from normal and asthmatic subjects.

Numbers of circulating basophils are increased in asthmatic subjects, compared to normal subjects. Basophil enriched cell preparations from normal and asthmatic subjects were challenged in vitro with the calcium ionophore A23187, anti-IgE, or opsonized zymosan to study leukotriene C4 formation, histamine release, and prostaglandin D2 formation. No prostaglandin D2 formation by basophils was observed. Furthermore, opsonized zymosan was not capable of inducing any mediator formation or release from basophils. At optimal stimulation conditions no differences were found between basophils from normal and asthmatic subjects concerning A23187 or anti-IgE induced leukotriene C4 formation or histamine release. A23187 and anti-IgE induced leukotriene C4 formation were in the range of 1-20 and 0.6-4.8 pmol/10(6) basophils respectively.     

Altered surface expression of CD11 and Leu 8 during human basophil degranulation.

Immunofluorescence and flow cytometric techniques have been used to study changes in surface Ag expression and viability that occur during human basophil degranulation. Treatment with polyclonal anti-IgE, FMLP, or the calcium ionophore A23187 induced histamine release, along with rapid and sustained unimodal increases in basophil CD11b mean fluorescence intensity. In contrast, treatment with anti-IgE or FMLP resulted in a decrease in Leu 8 expression. Degranulation did not significantly affect basophil viability (as determined by exclusion of propidium iodide), scatter characteristics, or percentage of identifiable IgE-bearing cells, and an inconsistent association was seen between percent histamine release and reduction in the percent of cells identified by light microscopy after staining with alcian blue. For anti-IgE, dose-dependent changes in CD11b, CD11c, and Leu 8 expression were seen (optimal at 0.1, 0.1, and 1 microgram/ml, respectively), although CD11a expression remained unchanged. Histamine release was optimal at 0.3 microgram/ml anti-IgE, and at superoptimal concentrations, reduced CD11b expression was observed which paralleled decreases in histamine release; reduction of the expression of Leu 8, however, occurred equally at optimal and superoptimal concentrations of anti-IgE. Kinetic analyses of these responses revealed that CD11b up-regulation proceeded more rapidly than histamine release, whereas Leu 8 down-regulation was much slower and did not plateau until 120 min of stimulation. Although changes in CD11b mean fluorescence intensity correlated with the magnitude of histamine release, exposure to stimuli in the absence of calcium (which blocked degranulation) resulted in similar alterations in CD11b and Leu 8, suggesting that degranulation was not required for changes in the surface expression of these adhesion molecules. Interestingly, pretreatment of basophils with drugs that either inhibited or enhanced histamine release (isobutylmethylxanthine and cyclosporin A vs cytochalasin B, respectively) significantly decreased the magnitude of anti-IgE-induced CD11b up-regulation; down-regulation of Leu 8 expression was also partially inhibited by treatment with isobutylmethylaxanthine. These studies demonstrate that activation of human basophils by secretagogues in vitro results in a variety of phenotypic changes including alterations in surface expression of adhesion molecules, and suggest that degranulation in vivo may be accompanied or preceded by changes in adhesion-related functions.     

The mechanism of mediator release from human basophils induced by platelet-activating factor.

Platelet-activating factor (PAF) is a lipid mediator able to induce a variety of inflammatory processes in human peripheral blood cells. We have investigated the effect of PAF on the release of chemical mediators from human basophils of allergic and normal donors. PAF (10 nM to 1 microM) caused a concentration-dependent, noncytotoxic histamine release (greater than or equal to 10% of total) in 27 of 44 subjects tested (24 atopic and 20 nonatopic donors). The release process was either very rapid (t1/2 approximately equal to 10 s) or quite slow (t 1/2 approximately equal to 10 min), temperature- and Ca2(+)-dependent (optimal at 37 degrees C and 5 mM Ca2+). Coincubation of PAF with cytochalasin B (5 micrograms/ml) enhanced the release of histamine induced by PAF and activated the release process in most donors (42 of 44). Atopics did not release significantly more histamine than normal subjects, and the percentage of PAF responders (greater than or equal to 10% of total) was nearly the same in the two groups. Histamine release was accompanied by the synthesis and release of leukotriene C4, although this lagged 1 to 2 min behind histamine secretion. Lyso-PAF (100 nM to 10 microM), alone or together with cytochalasin B, did not release significant amounts of histamine. The release of histamine activated by PAF was inhibited by the specific PAF receptor antagonist, L-652,731, with an IC50 of 0.4 microM. There was a partial desensitization to PAF when the cells were preincubated with PAF (100 nM to 1 microM) for 2 min in the absence of Ca2+, whereas the cells remained responsive to anti-IgE (0.1 micrograms/ml). If neutrophils were removed from the basophil preparation by a Percoll gradient or a countercurrent elutriation technique, there was a significant decrease in PAF-induced histamine release. PAF (1 microM) was able to induce a very rapid, transient rise (peak less than 10 s) in [Ca2+]i in purified basophils analyzed by digital video microscopy. Finally, among human histamine-containing cells, the basophils are unique in degranulating following a PAF challenge. Mast cells from human lung, skin, or uterus failed to respond to PAF (10 nM to 1 microM) regardless of the presence or absence of cytochalasin B (5 micrograms/ml). Our results demonstrate that PAF is able to induce the release of inflammatory mediators from human basophils, and that neutrophils can influence this response. It is suggested that PAF-induced basophil activation can play a role in the pathogenesis of allergic disorders.     

Cholinergic-induced [3H] noradrenaline release in rat brain cortical slices is mediated via a pertussis toxin sensitive GTP binding protein and involves activation of protein kinase C

The involvement of a GTP-binding protein (G-protein) in the process of neurotransmitter release was examined using pertussis toxin and cholera toxin. Cholinergic agonists are shown to mediate [3H]noradrenaline release in rat brain slices via a pertussis toxin (1.2 micrograms/ml) sensitive, and cholera toxin (0.5 microgram/ml) insensitive G-protein. An indication for the involvement of a G-protein and phospholipase C activation in the release process was implied from the inhibitory effect of neomycin on K+-, veratridine- and carbachol-induced-norepinephrine release. Depolarizing agents mediate a neomycin-sensitive release, which is not which is not affected either by pertussis toxin or cholera toxin, suggesting a different mode of phospholipase C activation, unlike carbachol-induced release, which is both neomycin and pertussis toxin sensitive. Similarly, a hormone-sensitive carrier activated by phenylephrine not via alpha 1-adrenergic receptors, mediates a non-exocytosis efflux which is not affected by neomycin and is shown to be pertussis toxin-insensitive. The inhibitory action of protein kinase C inhibitors polymyxin B, K252a and H-7 [(1-(5-isoquinolinesulphonyl)-2-methyl-piperazine] on release, strongly suggests its participation in the process. Polymyxin B, a relatively selective protein kinase C inhibitor, inhibited carbachol-induced release (IC50 = 0.53 microM) as well as the K+ and the veratridine induced [3H] noradrenaline release, K252a, an inhibitor of various protein kinases at the ATP site, and H-7, another protein kinase C inhibitor, inhibited carbachol-induced noradrenaline released with IC50 = 35 nM and 3 microM respectively. Consistent with its inability to activate phospholipase C, phenylephrine-induced noradrenaline efflux was unaffected by polymyxin B (greater than 70 microM). These results offer more supportive evidence for a major role played by the dual messengers inositol trisphosphate and diacylglycerol (IP3/DG) in the mechanisms of neuronal release.     

Mechanisms of action of cyclosporin A on islet alpha- and beta-cells. Effects on cAMP- and calcium-dependent pathways.

The involvement of cAMP- and calcium-dependent pathways on the inhibitory effect of CsA (0.5 micrograms/ml) on insulin and glucagon release was studied in collagenase-isolated islets. CsA suppressed by 50% the release of insulin in pertussis toxin treated islets stimulated by 20 mM D-glucose. CsA blocked glucagon and insulin release induced by 0.2 mM IBMX (80% and 50% respectively). Similarly it inhibited glucagon and insulin release induced by 1 microM A23187 (53% and 40% respectively). CsA also abolished 0.1 microM glucagon-induced insulin release and 10 ng/ml VIP-induced glucagon release (70% and 38% respectively). The glucagon response to 2 mM D-glucose and to 10 mM arginine was decreased 25% and 45% respectively by CsA. The inhibitory effect of 0.1 microM somatostatin on insulin release was significantly abolished by CsA (p less than 0.001 vs control). On the other hand 1 microM forskolin induced insulin and glucagon release was not modified by CsA. Rats treated with CsA (10 mg/kg body wt) during 10 days showed hyperglycaemia, hypoglucagonemia and higher contents of pancreatic glucagon. It is concluded that CsA affects alpha- and beta-cell function, in vivo and in vitro, acting through calcium and cAMP-dependent pathways. This latter pathway involves the Ca(2+)-calmodulin dependent phosphodiesterase and the regulatory proteins Gs and Gi.     

Human granulocyte-macrophage colony-stimulating factor and other cytokines prime human neutrophils for enhanced arachidonic acid release and leukotriene B4 synthesis

Human recombinant granulocyte-macrophage CSF (GM-CSF) "primes" neutrophils for enhanced biologic responses to a number of secondary stimuli. Here, we examined the properties of neutrophil priming by GM-CSF and other growth factors such as human rTNF and granulocyte CSF. Although GM-CSF has a negligible direct effect on [3H]arachidonic acid release, it enhances or "primes" neutrophils for three- to fivefold increased release of [3H]arachidonic acid, induced by 1.0 microM A23187 and the chemotactants FMLP, platelet-activating factor, and leukotriene B4 (LTB4) (all 0.1 microM). The priming effects of GM-CSF were concentration- and time-dependent (maximum 100 pM, 1 h at 23 degrees C), and consistent with the determined dissociation constant of the human GM-CSF receptor. Indomethacin (10(-8) M), cycloheximide (100 micrograms/ml), and pertussis toxin (200 ng/ml, 2 h at 37 degrees C) had no effect on GM-CSF-, A23187, or platelet-activating factor-induced [3H]arachidonic acid release. The lipoxygenase inhibitor, nordihydroguaiaretic acid, however, totally abolished A23187-induced [3H]arachidonic acid release from both diluent- and GM-CSF-treated neutrophils. Consistent with this observation, we found that GM-CSF-pretreated neutrophils synthesize increased levels of LTB4 after stimulation with A23187 and chemotactic factors. GM-CSF enhances neutrophil arachidonic acid release and LTB4 synthesis, and thereby may amplify the inflammatory response to chemotactic factors and other physiologically relevant stimuli.     

cAMP-independent effects of cholera toxin on B cell activation. I. A possible role for cell surface ganglioside GM1 in B cell activation

Cholera toxin has been used as a tool to study the effects of cAMP on the activation of B cells but may have effects independent of its ability to elevate cAMP. We found five lines of evidence which suggested that cholera toxin suppressed mitogen-stimulated B cell activation through a cAMP-independent pathway. 1) Cholera toxin (1 microgram/ml) was consistently more suppressive than forskolin (100 microM) despite the induction of higher intracellular cAMP levels by forskolin. 2) Cholera toxin was more suppressive at 1 microgram/ml than at 0.1 microgram/ml despite equivalent elevations of cAMP. 3) Washing B cells following their incubation with cholera toxin reversed much of the inhibition without altering intracellular cAMP levels. 4) The A subunit of cholera toxin, which at high concentrations (10 micrograms/ml) induced levels of cAMP comparable to those induced by cholera toxin (1 and 0.1 microgram/ml), did not inhibit B cell activation. 5) cAMP derivatives at high concentrations were much less effective than was cholera toxin in suppressing B cell activation. Although the elevation of cAMP may cause a mild inhibition of B cell proliferation, we found that even a marked elevation of cAMP did not suppress B cell proliferation, unless the elevation was persistent. We did, however, observe that the degree of toxin inhibition more closely paralleled binding of the toxin to B cells than toxin stimulation of cAMP. This result raised the possibility that binding of cholera toxin to its ganglioside GM1 receptor mediated an inhibitory signal which suppressed B cell proliferation.     

Signal transduction events in human basophils. A comparative study of the role of protein kinase C in basophils activated by anti-IgE antibody and formyl-methionyl-leucyl-phenylalanine.

We have compared the transmembrane signals generated in human basophils by two distinct stimuli, anti-IgE antibody and FMLP (f-met peptide). Although both stimuli resulted in the activation of protein kinase C (PKC) and an increase in intracellular free calcium, there were substantial differences between the two which suggested that distinct signal transduction mechanisms were operating. We have confirmed an earlier observation that the cross-linking of IgE led to an increase in membrane PKC activity with no apparent concomitant loss of cytosolic PKC and established that in contrast, the univalent stimulus, f-met peptide, resulted in the canonical translocation of cytosolic PKC to the membrane. Furthermore, unlike anti-IgE-stimulated basophils, there was no clear relationship between the increase in PKC activity and the subsequent release of histamine. Two PKC inhibitors, staurosporine (0.1 to 1 nM) and sphingosine (25 to 50 microM), inhibited anti-IgE induced release, yet, potentiated the release of mediators after a challenge with 1 microM f-met peptide. Both stimuli led to an increase in the intracellular Ca2+ levels that correlated well with the release of histamine, however, the anti-IgE-induced responses were typically only 50% of those required to give equivalent histamine release when f-met peptide initiated release. Pharmacologic evidence suggested that the up-regulation of PKC was required for a full IgE-mediated Ca2+ response and that PKC contributed to the elevated Ca2+ levels that persist for up to 15 min after the addition of anti-IgE. In contrast, the PKC inhibitor, staurosporine, did not affect the initial increase in Ca2+ after the addition of f-met peptide but reduced the rate at which Ca2+ was removed from the cytosol. Experiments with the phorbol ester, PMA, suggested that substantial degranulation can occur in the absence of any increase in intracellular Ca2+. The addition of 10 ng/ml PMA 10 min before the addition of f-met peptide did not affect the magnitude of the initial Ca2+ transient but increased the rate at which Ca2+ levels returned to a stable baseline. Similar pretreatment with PMA almost completely abolished the anti-IgE antibody-induced Ca2+ response. These experiments, together with other previous data, suggest that the activation of PKC is a prodegranulatory component of the IgE-mediated signal transduction pathway, yet serves principally to modulate the Ca2+ signal when f-met peptide initiates release.     

Calcium-dependent regulation of progesterone production by isolated rat granulosa cells: effects of the calcium ionophore A23187, prostaglandin E2, dl-isoproterenol and cholera toxin

The role of calcium in the regulation of ovarian steroidogenesis was investigated in granulosa cells from estradiol-treated immature rats. Incubation of granulosa cells with various calcium channel blockers (verapamil, cobalt or manganese) and a calcium chelator (EGTA) resulted in marked decreases in progesterone production in response to follicle-stimulating hormone (FSH), cholera toxin, prostaglandin E2, dl-isoproterenol and dibutyryl cyclic AMP (Bt2cAMP). Cyclic AMP production, however, was unaffected by treatment with EGTA and verapamil at concentrations which attenuated steroidogenesis (0.1-1.0 mM and 125 microM, respectively). Two inhibitors of the calcium-dependent regulatory protein, calmodulin [trifluoperazine, 40 microM and 1[bis-(p-chlorophenyl)methyl] 3-[2,4-dichloro-beta-(2,4- dichlorobenzyloxy )-phenethyl]imidazolium chloride, ( R24571 ) 20 microM] significantly inhibited both cyclic AMP and progesterone production elicited by these stimulatory agents. Over the concentration range of 62.5 ng/ml-1.0 micrograms/ml, the calcium ionophore A23187 increased basal progesterone production in a dose-dependent manner, with half-maximal stimulation at approximately 0.14 microgram/ml. Maximal steroidogenic response to the calcium ionophore (1 microgram/ml) however, was only 50% of that evoked by FSH (0.33 microgram/ml). A23187 (0.5 microgram/ml) significantly enhanced progesterone production stimulated by a low concentration of FSH (0.025 microgram/ml) but failed to potentiate the maximally stimulatory action of the gonadotropin (0.33 microgram/ml). These findings support our earlier suggestion that the calcium-calmodulin system plays a central role in the gonadotropic regulation of ovarian steroidogenesis and suggest that a transmembrane flux of extracellular calcium may be an important and common step in the mechanism of stimulation of granulosa cell progesterone production.     

Measurement of IgE on human basophils: relation to serum IgE and anti-IgE-induced histamine release.

The number of IgE molecules bound to human basophils was calculated from direct measurements of the IgE dissociated after exposing leukocytes to pH 3.7 acetate buffer in the cold. In 18 donors studied, cell-bound IgE ranged from 4000 to 500,000 molecules/basophil and correlated with the serum IgE concentration (r = 0.89, p less than 0.001) which ranged from 5 to 3,000 ng/ml. Sensitivity of these cells to anti-IgE was tested to explore the relationship between cell-bound IgE and the concentration of anti-IgE required for histamine release. Cells from some nonatopic donors (4000 to 100,000 IgE molecules/basophil) were as sensitive as cells from allergic donors (100,00 to 500,000 IgE molecules/basophil). Moreover, cells from donors having approximately the same cell-bound IgE concentration varied widely in their sensitivity to anti-IgE. We conclude that an intrinsic property of human basophils ("releasability") is an important parameter in determing mediator release.