Secretion Of Granule Constituents Research Articles

Differential effects of non-selective and selective phosphodiesterase inhibitors on human eosinophil functions.

1. The effect of non-selective (3-isobutyl-1-methylxanthine, IBMX; theophylline) and type IV- or type III/IV-selective (rolipram, RP 73401; zardaverine, tolafentrine) phosphodiesterase (PDE) inhibitors on human eosinophil functions was investigated. 2. For this purpose human eosinophils were purified from blood of healthy donors by a magnetic cell separation (MACS) technique to a purity > or = 99%. From the stimuli investigated (complement C5a; N-formyl-methionyl-leucyl-phenylalanine, fMLP; platelet activating factor, PAF; opsonized zymosan) C5a was selected to test the influence of the above mentioned compounds on secretion of granule constituents (eosinophil cationic protein, ECP; eosinophil-derived neurotoxin, EDN) as well as on formation of reactive oxygen species measured by luminol-enhanced chemiluminescence in intact cells. For comparison, inhibition of PDE IV activity in the cytosol of disrupted cells, which contains about 75% of total PDE IV activity, was determined. 3. Both theophylline and IBMX inhibited the two cell responses with IC50 values which were in the range of their IC50 values obtained for inhibition of PDE IV activity in the cell-free system. The beta 2-adrenoceptor agonist, salbutamol (1 mumol l-1), which by itself did not substantially influence the two cell responses, only marginally improved the potency of theophylline and IBMX in inhibiting ECP/EDN secretion. Only the IC50 value of IBMX for inhibition of chemiluminescence was lowered by about one order of magnitude in the presence of salbutamol. 4. In contrast, none of the selective PDE inhibitors tested substantially inhibited the two cell responses at concentrations up to 10 mumol l-1. This was surprising because all of the compounds investigated inhibited PDE IV activity in the cell-free system with IC50 values which were at least 30 fold lower than the highest concentration of the compounds used with intact cells. In combination with salbutamol, however, both ECP/EDN secretion and chemiluminescence was inhibited by rolipram and zardaverine with IC50 values similar to the IC50 values for inhibition of PDE IV activity. Although RP 73401 and tolafentrine also inhibited both cell responses in the presence of salbutamol, the potency of these two compounds in inhibiting eosinophil function in intact cells was at least two orders of magnitude lower than would have been expected from the inhibition of PDE IV activity in the cell-free system. 5. These results indicate that (i) C5a-stimulated human eosinophils are sensitive to inhibition by then on-selective PDE inhibitors theophylline and IBMX, (ii) the inhibitory effect of these non-selective PDE inhibitors cannot be mimicked by selective PDE IV or PDE III/IV inhibitors although human eosinophils almost exclusively contain PDE IV; (iii) the selective PDE inhibitors need an additional cyclic AMP trigger like a beta 2-adrenoceptor agonist to be effective; but (iv) under the latter conditions inhibition of cell responses in intact cells does not correspond to inhibition of PDE IV activity in the cell-free system.6. We conclude that the non-selective PDE-inhibiting xanthines may inhibit C5a-stimulated human eosinophil responses by other action(s) in addition to PDE IV inhibition, and that inhibition of PDE IV activity in the cell-free system by the selective inhibitors may not generally represent the potency of the compounds in intact cells.

Characterisation of ovine mast cells derived from in vitro culture of haemopoietic tissue

Ovine mast cells generated in vitro from bone marrow (BMMC) were compared with mucosal mast cells (MMC) isolated from parasitised abomasum. Ultrastructurally, the granules of BMMC were partially developed and immature. Both cell types contained β-hexosaminidase, arylsulfatase, histamine, dopamine and sheep mast cell proteinase (SMCP). Greater amounts of β-hexosaminidase, but less SMCP, histamine and arylsulfatase were present in BMMC. Stimulation with calcium ionophore A23187 caused the secretion of granule constituents and generation of leukotriene C 4 by BMMC in a dose-dependent manner. An additional [ 3H]diisopropylfluorophosphate-binding 31 500 mol. wt. serine esterase, antigenically related to SMCP (27 000 mol. wt.) was present in cultures of BMMC but was not detected in isolated MMC. Both enzymes were detected in BMMC by Day 7 of culture and were secreted concomitantly following stimulation of BMMC with ionophore.

Histamine and its antagonists in asthma

Histamine has been recognized as a potent effector agent in allergic responses for many years. Windaus and Vogt’ first synthesized histamine in 1907, but it was Dale and Laidlaw who originally suggested that this substance could be liberated during an anaphylactic reaction when they observed that the features of anaphylaxis could be reproduced by the intravenous injection of histamine. Best et a1.3 later demonstrated that high concentrations of histamine were present in lung tissue. The association of histamine with asthma came when Weis et a1.4 found that intravenous injection of the amine in subjects with asthma induced wheezing. Further circumstantial evidence linking histamine and asthma came when blood from subjects with asthma was demonstrated to release histamine when blood was challenged with specific antigen in vitro,‘. 6 Subsequently, several authors7. * found increased plasma histamine concentrations in subjects with asthma during exacerbation of asthma. In 1951 Schild et a1.9 described the release of histamine from lung tissue after allergen challenge and commented that this coincided with the bronchoconstriction that was induced. Histamine is found in human mast cells and basophils” and is stored in secretory granules in association with heparin proteoglycan” and a variety of other preformed mediators. In Ishizaka’~‘~ classical description of mast cell activation, histamine and the remaining granule constituents are released when antigen interacts with specific IgE FcRl receptors on the surface of the mast cell. This results in the stimulation of membrane phospholipid metabolism, followed by influx of extracellular and mobilization of intracellular calcium. This intracellular signal results in the secretion of granule constituents and the mobilization of arachidonic acid from membrane phospholipid stores. Although several authors found elevations of

The kinetics of secretion from permeabilized human neutrophils: release of elastase and correlations with other granule constituents and right angle light scatter.

We have previously reported that human neutrophils can be permeabilized with the cholesterol-complexing agent digitonin. These permeabilized cells can be induced to secrete lysosomal constituents when exposed to micromolar levels of free Ca2+, a process that is enhanced by certain guanine nucleotides. We examined the kinetics in this system by employing both direct and indirect measures of secretion. A continuous, fluorescent assay of elastase permits real-time monitoring of secretion from azurophil granules. The kinetics of elastase release proved to be rapid, beginning within 3-10 sec and reaching a maximum at 1-2 min. Changes in the Ca2+ concentration did not affect the "lag period" for release. A comparison of the Ca2+ dose-response curves for release of the various granule constituents indicated that elastase was being secreted along with other contents of the azurophil granules. Changes in right angle light scatter (RLS), which have been shown to correlate closely with secretion, also commenced rapidly after the addition of Ca2+; when measured simultaneously, both the Ca2+ dose-response characteristics for changes in RLS and elastase release were very similar. Changes in RLS could be halted within 5 sec by excess EGTA and restarted promptly by repletion with secretory concentrations of Ca2+. In addition, neomycin, a phospholipase C inhibitor, profoundly diminished degranulation as monitored by RLS and end-point techniques. A continuous assay employing 9-aminoacridine self-quenching as a measure of secretion proved far less satisfactory, but, nonetheless, produced similar kinetics and dose-response characteristics.(ABSTRACT TRUNCATED AT 250 WORDS)

Malonaldehyde formation in intact platelets is catalysed by thromboxane synthase.

Imidazole and compound L8027 (selective inhibitors of thromboxane synthase) produced parallel inhibition of malonaldehyde and thromboxane B2 secretion induced by collagen or thrombin in gel-filtered suspensions of human platelets. Comparing the effects of these inhibitors and aspirin on secretion of granule constituents indicated that platelet degranulation depends mainly on thromboxane production; prostaglandin endoperoxides contributed little.