Effects of nedocromil sodium on in vitro induced migration, activation, and mediator release from human granulocytes.

Using the allergen-induced late-phase asthmatic reaction as a working model, we studied the activity of certain inflammatory cells and their reaction to nedocromil sodium. The processes that were examined in vitro included the following: the chemotaxis of purified neutrophils and eosinophils, the early steps of neutrophil and eosinophil activation, and the release of mediators from these cells. Nedocromil sodium strongly inhibited neutrophil mobilization caused by four chemotactic factors (zymosan activated serum, N-formyl-methionyl-leucyl-phenylalanine platelet-activating factor [PAF], and leukotriene B4 [LTB4] and eosinophil mobilization caused by two factors (PAF and LTB4). In vitro treatment of eosinophils from normal subjects with picomolar concentrations of interleukin-3, interleukin-5, or granulocyte-macrophage colony stimulating factor increased the chemotactic responsiveness toward PAF and LTB4 and induced a chemotactic responsiveness toward N-formyl-methionyl-leucyl-phenylalanine and neutrophil activating factor/interleukin-8. The zymosan activated serum-induced chemotactic responsiveness remained unaltered. Nedocromil sodium inhibited the cytokine-primed chemotactic responsiveness to the various chemotaxins, not the influence of the cytokines on the cells. Activation of granulocytes, as measured by Ca2+ influx, was not inhibited by nedocromil sodium. Mediator formation in eosinophils was modified only slightly. These results suggest that inhibiting the mobilization of inflammatory cells in the lung tissue may be an important action of nedocromil sodium. Therefore these effects may be relevant to the treatment of asthma given the role of airway inflammation in this disease process.

Nedocromil sodium inhibits the A23187- and opsonized zymosan-induced leukotriene formation by human eosinophils but not by human neutrophils.

1. Inflammatory cells such as eosinophils and neutrophils are thought to contribute actively to the pathogenesis of asthma by the release of bronchoconstrictor mediators including leukotrienes. Previous studies have revealed the almost exclusive synthesis of leukotriene C4 (LTC4) by human eosinophils and of leukotriene B4 (LTB4), 20-OH-LTB4 and the non-enzymatically formed LTB4-isomers by neutrophils when stimulated in vitro with the calcium ionophore A23187 or opsonized zymosan (OZ). In this study we have investigated whether nedocromil sodium, a new anti-asthma drug, was capable of inhibiting A23187- and OZ-induced leukotriene formation by these cells. 2. Nedocromil sodium inhibited A23187- and OZ-induced LTC4 formation by eosinophils in a concentration-dependent manner (mean IC30 for A23187: 5.6 X 10(-5) M; mean IC30 for OZ: 6.3 X 10(-5) M), whereas it did not inhibit A23187- and OZ-induced LTB4 formation by neutrophils. 3. Extension of the preincubation time of the cells with the drug did not alter the observed inhibitory capacity. The optimal preincubation time was 5 min. 4. The in vitro inhibition of LTC4 formation by eosinophils by nedocromil sodium may be a valuable property of this drug in the treatment of asthma.

Inhibitory effects of nedocromil sodium on the in vitro induced migration and leukotriene formation of human granulocytes.

Inflammatory cells, such as neutrophils and eosinophils, are thought to actively contribute to the pathogenesis of asthma since they infiltrate into the lung tissue and may be activated locally to release bronchoconstrictor mediators. In this study we provide evidence that nedocromil sodium is capable of effectively inhibiting the platelet-activating factor (PAF) and zymosan-activated serum (ZAS)-induced chemotaxis of polymorphonuclear granulocytes (PMN) [IC50 approximately 1 nmol/L and 0.1 mumol/L respectively]. The same inhibitory potency was obtained with sodium cromoglycate. Thus, nedocromil sodium may effectively inhibit the mobilisation of inflammatory cells in the lung. Furthermore, nedocromil sodium is capable of inhibiting the formation of the bronchoconstrictor mediator leukotriene-C4 (LTC4) by eosinophils in a concentration-dependent way [IC30 for A23187: 5.6 10(-5) mol/L; IC30 for opsonised zymosan (OZ): 6.3 10(-5) mol/L], whereas this drug is not capable of inhibiting leukotriene-B4 (LTB4) formation by neutrophils. These findings indicate that nedocromil sodium inhibits the release of bronchoconstrictor mediators not only from mast cells but also from eosinophils.

Leukotriene C4 formation by purified human eosinophils can be induced by arachidonic acid in the absence of calcium-ionophore A23187.

Addition of arachidonic acid (50 microM) to purified human eosinophils leads to the formation of considerable amounts of LTC4 [11.3 +/- 1.3) x 10(6) molecules/cell, mean +/- SEM, n = 10), 15-HETE [412 +/- 142) x 10(6) molecules/cell, mean +/- SEM, n = 3) and 15-series leukotrienes [35 +/- 15) x 10(6) molecules/cell, mean +/- SEM, n = 3). The ratio of the amounts of LTC4 and 15-lipoxygenase products was found to be strongly dependent on the arachidonic acid concentration, being relatively large at low arachidonic acid concentrations and very small at high arachidonic acid concentrations. Platelet activating factor (1 microM) was able to enhance significantly the production of LTC4 but not that of 15-lipoxygenase products. As arachidonic acid was found to be capable of inducing a fast, transient rise in the cytosolic free Ca2+ concentration, this explains, at least partly, its ability to induce the Ca2+-dependent formation of LTC4.

An improved method for the isolation of eosinophilic granulocytes from peripheral blood of normal individuals.

A simple and improved procedure is described for the isolation of human eosinophils from normal individuals with about 2% eosinophils in their peripheral blood. This method comprises a preincubation of a mixed granulocyte preparation with 10 nM fMLP for 10 min at 37 degrees C followed by a one-step density centrifugation on isotonic Percoll. The recovery of eosinophils is 49 +/- 4% at 89 +/- 4% purity. Because of the relatively high rate of recovery, it is now possible to isolate eosinophils from blood samples as small as 20 ml. Because treatment with fMLP may alter the functional activity of the eosinophils, the following metabolic functions were tested: changes in cytosolic free Ca2+, oxygen consumption, chemiluminescence, chemotaxis, and leukotriene C4 formation. We found that 10 nM fMLP does not activate eosinophils in these assays, whereas 1 microM fMLP does (with the exception of chemotaxis). Furthermore, pretreatment of eosinophils with 10 nM fMLP did not influence the response to other stimuli in these assays. The usefulness of this method was evaluated by comparing it with three other previously described procedures. In our hands, only the method presented here enabled us to isolate eosinophils from normal individuals with about 2% eosinophils in their peripheral blood.

Arachidonic acid can induce leukotriene C4 formation by purified human eosinophils in the absence of other stimuli.

Stimulation of purified human eosinophils with 50 microM arachidonic acid leads to the production of leukotriene C4, 15-hydroxy-eicosatetraenoic acid and 15-series leukotrienes. The ratio of the amounts of leukotriene C4 and 15-lipoxygenase products was found to be strongly dependent on the arachidonic acid concentration, being relatively large at low arachidonic acid concentrations and very small at high arachidonic acid concentrations. In the presence of 1 microM platelet-activating factor a significant elevation of leukotriene C4 formation is observed, whereas the formation of 15-lipoxygenase products remains unaltered. As arachidonic acid was found to be capable of inducing a fast, transient rise in the cytosolic free Ca2+ concentration, this explains at least partly its ability to induce the Ca2+-dependent formation of leukotriene C4.

Terbutaline sustained-release treatment during one week 5 mg b.i.d. compared to one week placebo: terbutaline plasma levels, c-AMP plasma levels, lung function and tremor measurement.

In a group of ten patients with chronic asthmatic bronchitis, a good improvement of the lung function was achieved by a sustained-release terbutaline preparation (Bricanyl Retard) in a dosage of 5 mg twice daily. Tremor measurements and c-AMP plasma level showed a statistically significant increase. A good patient compliance was achieved with this dosage of terbutaline two times daily. The sustained-release preparation caused a rather constant and low terbutaline plasma level. We think that this relative low terbutaline plasma level is the most important reason for the lack of side effects.

Platelet-activating factor induces leukotriene C4 synthesis by purified human eosinophils.

Platelet-activating factor, at a concentration of 10 microM, was capable of inducing leukotriene C4 synthesis by eosinophils of healthy donors, i.e. (3.1 +/- 0.3) x 10(6) molecules leukotriene C4/cell (n = 31, mean +/- SEM, cell purity 87 +/- 2%). Reversed-phase high performance liquid chromatography analysis demonstrated the exclusive synthesis of leukotriene C4. At a concentration of 1 microM, platelet-activating factor was capable of significantly enhancing the calcium ionophore A23187, the opsonized zymosan or the arachidonic acid induced leukotriene C4 synthesis by eosinophils. These results show that PAF is capable of inducing and enhancing the leukotriene C4 formation by human eosinophils.

Blood lymphocyte subpopulations in extrinsic and intrinsic asthmatics.

Blood leukocyte numbers and proportions of T lymphocyte subsets were studied in extrinsic asthmatics (EA), intrinsic asthmatics (IA), IA systemically treated with corticosteroids (IA + C), and in age-matched control subjects. The EA and IA showed an increased number of eosinophils. During corticosteroid therapy of IA, the eosinophil number remained elevated, whereas there was a slight decrease in the number of circulating lymphocytes. The proportion of T cells of the suppressor/cytotoxic phenotype carrying the Leu-2a antigen was significantly lower in the IA than in all other groups. In the IA + C group, the proportions of Leu-3a/3b and Leu-2a positive T lymphocytes returned to normal, although the patients still exhibited asthmatic symptoms. These findings suggest that cellular immunologic factors might be involved in the pathogenesis of intrinsic asthma.

Lung function improvement, tremor measurements and c-AMP determinations in a group of ten patients with asthmatic bronchitis after one week sustained release theophylline medication (theophylline plasma level +/- 10 mg/l) compared to one week placebo.

After one week theophylline therapy with a sustained release preparation (TheoDur 200 and 300 mg), a mean theophylline level of 10 mg/l was obtained in a group of ten patients with chronic asthmatic bronchitis with a mean dosage of 760 mg theophylline/24 h. A good improvement of the lung function was achieved, although only statistically significant for the mid maximal expiratory flow rate (MMEF) and the end expiratory specific airway conductance (sGaw) compared to placebo therapy. Tremor measurements and cyclic-AMP determinations showed a slight increase after theophylline therapy compared to placebo, although not statistically significant.

Pharmacodynamics (lung function tests, tremor measurements and cAMP determinations) of a single dose of 0.5 mg terbutaline subcutaneously during sustained-release theophylline medication in patients with asthmatic bronchitis.

A group of 10 patients with chronic asthmatic bronchitis was titrated with slow-release theophylline (Theolin Retard) to a steady state plasma theophylline level of 10 mg/l. After one week of this treatment a single s.c. dose of 0.5 mg terbutaline (Bricanyl) at 10:30 a.m was administered. Lung function at 8:15 a.m. on this day was better than on the reference day without any medication, but the differences were not statistically significant. At 11:00 p.m., 30 min after s.c. administration of terbutaline all lung function parameters (VC, FEV1, MMEF and sGaw) were significantly raised compared to the values of 8:15 p.m. cAMP levels and tremor were significantly higher after the combined medication (30, as well as 150 min after s.c. administration of terbutaline) than on the reference day. This observation implies that we have to be careful with the administration of terbutaline s.c. to patients on theophylline treatment in the daily practice of the lung function laboratory.

Exclusive leukotriene C4 synthesis by purified human eosinophils induced by opsonized zymosan.

Purified human eosinophils were challenged with N-formyl-methionyl-leucyl-phenylalanine, leukotriene B4, platelet-activating-factor, valyl-glycyl-seryl-glutamic acid, phorbol myristate acetate, zymosan, opsonized zymosan and the calcium ionophore A23187 to induce leukotriene synthesis. Reversed-phase high performance liquid chromatography analysis demonstrated the almost exclusive synthesis of leukotriene C4 by eosinophils of 11 healthy donors after challenge with opsonized zymosan [(22 +/- 4) X 10(6) molecules LTC4/cell, mean +/- SE] or the calcium ionophore A23187 [(54 +/- 7) X 10(6) molecules LTC4/cell, mean +/- SE]. The other agents were not capable of inducing leukotriene formation. When in addition to opsonized zymosan N-formyl-methionyl-leucyl-phenylalanine or platelet-activating factor were added a significant increase of the leukotriene C4 synthesis by eosinophils was observed. These results suggest that eosinophils might be triggered to produce considerable amounts of the spasmogenic leukotriene C4 in vivo by C3b- and/or IgG-mediated mechanisms e.g. phagocytosis.

Pharmacokinetics and pharmacodynamics of a short- and long-acting theophylline medication (Theolair and Theolair retard) in normals and patients with COLD. Part III: Plasma cyclic AMP.

Supporting evidence was found for the beta-blockade theory of Szentivanyi. Asthmatics (n = 6) showed lower basal plasma cyclic AMP levels and lower cyclic AMP levels after administration of a short- and long-acting theophylline preparations (Theolair and Theolair retard) than normals (n = 6) did. No difference was found in this respect between Theolair and Theolair retard. Basal cyclic AMP levels showed a diurnal variation, with highest levels in the morning which gradually decreased during the day. The correlation of the plasma cyclic AMP levels with the lung function change after stimulation with theophyllines in asthmatics was rather poor. This is probably caused by the rather low changes in plasma cyclic AMP levels.

Desensitization of the beta-adrenergic receptor on leucocytes after long-term oral use of a beta-sympathicomimetic; its effect on the beta-adrenergic blockade hypothesis of Szentivanyi.

Development of beta-receptor desensitization in leucocytes of asthmatics and normals during oral therapy with terbutaline was established by means of in vitro stimulation with terbutaline and isoproterenol. This desensitization disappeared about one week after oral therapy was discontinued. In addition no difference in stimulation pattern, measured as increase in intracellular c-AMP content, was determined between asthmatics and normals when both were not receiving therapy. Even when the asthmatics showed strong bronchial hyperreactivity no difference was found. These results do not support the beta-adrenergic blockade hypothesis of Szentivanyi.

Defective erythrocyte pyruvate kinase.

A defective pyruvate kinase (EC 2.7.1.40) is described. The abnormal PK is characterized by a shift in the R in equilibrium T equilibrium to the T-state. The Ko.5 for the substrate phosphoenol pyruvate is about 6 times higher than for the normal enzyme, while the KM value for the positive effector Fru-1, 6-P2 is increased. In agreement with a shift to the T-state is the increased affinity of the abnormal enzyme for the negative effectors ATP and alanine. The results are discussed in relation to other abnormal pyruvate kinases.