Human Lung Fragments Research Articles

The inhibition by azatadine of the immunological release of leukotrienes and histamine from human lung fragments

The effects of the H 1 antihistamine azatadine on the antigen-induced release of slow reacting substance of anaphylaxis (leukotrienes C 4 and D 4) and histamine from passively sensitized human lung fragments were determined. Azatadine 0.1–10 μM significantly and dose dependently inhibited the release of leukotrienes from lung by 22–71%. Histamine release was also inhibited by azatadine 10 μM.

Chloroquine inhibits leukotriene release from human and guinea-pig lung fragments

Chloroquine inhibits leukotriene release from human and guinea-pig lung fragments

Characterization of the adenosine receptor responsible for the inhibition of histamine and SRS-A release from human lung fragments.

The inhibitory effects of a range of natural and synthetic derivatives of adenosine on the antigen-induced release of histamine and slow reacting substance of anaphylaxis (SRS-A) from human lung has been studied. The nucleotides ATP, ADP and AMP appear to act by being converted to adenosine. The rank order of inhibitory potency of the synthetic analogues indicates that these compounds act at an extracellular A2/Ra purinoceptor. The xanthines, 1, 3-diethyl-8-phenylxanthine, 8-phenyltheophylline and theophylline antagonized the inhibitory action of N-ethyl-carboxamideadenosine competitively. Theobromine was inactive. This supports the view that the inhibitory receptor is of the A/R type. Hexobendine and dipyridamole, reported to antagonize the uptake of adenosine, failed to modify the response of human lung fragments to adenosine. The P site agonist 2',5' dideoxyadenosine inhibited the release of histamine and SRS-A. This effect was not prevented by the inhibitors of uptake, hexobendine and dipyridamole, nor was it antagonized by 8-phenyltheophylline.

PROCEEDINGS OF THE AUSTRALASIAN SOCIETY OF CLINICAL AND EXPERIMENTAL PHARMACOLOGISTS

PROCEEDINGS OF THE AUSTRALASIAN SOCIETY OF CLINICAL AND EXPERIMENTAL PHARMACOLOGISTS

BRL 22321, a compound that has mast cell stabilizing activity similar to that of disodium cromoglycate and in addition has smooth muscle relaxing activity.

BRL 22321 is 4,9-dihydro-6,7-dimethyl-4,9-dioxo-1H-naphtho[2,3-d]-v-triazole sodium salt. It combines some of the chemical structural features of disodium cromoglycate (DSCG) and theophylline. It has mast cell stabilizing activity similar to that DSCG and in addition it has some smooth muscle relaxant activity. BRL 22321 was more potent than DSCG as an inhibitor of rat passive cutaneous and peritoneal anaphylaxis (PCA and PPA) and histamine release by antigen from passively sensitized rat peritoneal cellsin vitro. In the rat PCA test it was active when given orally and a large intravenous dose of DSCG reduced the potency of subsequent intravenous doses of either DSCG or BRL 22321, suggesting that the compounds were active by a common pathway. The compounds had similar potencies as inhibitors of the release, by antigen, of histamine and slow reacting substance of anaphylaxis (SRS-A) from passively sensitized human lung fragments. BRL 22321 was more potent than DSCG and theophylline as an inhibitor of a cyclic adenosine and a cyclic guanosine monophosphate phosphodiesterase. BRL 22321 inhibited histamine release in systems in which DSCG was inactive, for example it inhibited histamine release by antigen from chopped lung taken from hyperimmune guinea pigs and by anti-IgE from human leucocytes; in these tests it was more potent than theophylline. BRL 22321 had smooth muscle relaxant activity not shown by DSCG. It relaxed histamine elevated tone in guinea pig lung strip and tracheal spiral preparations at doses at which theophylline produced a dose dependent response. It was difficult to compare potencies since there was a difference in the nature of the responses to the drugs. DSCG had little activity over the same dose range or at higher doses. BRL 22321 also reduced increased airways resistance in the anaesthetized guinea pig produced by 5-hydroxytryptamine (5-HT) or histamine but at somewhat higher doses than those at which theophylline was active. BRL 22321 was not an antagonist of acetylcholine, histamine, 5-HT or SRS-A on the isolated guinea pig ileum.

Effects of dexamethasone on mediator release from human lung fragments and purified human lung mast cells.

Purified human lung mast cells released histamine, leukotrienes, prostaglandin (PG) D2, thromboxane B2 (TxB2), and PGF2 alpha in response to anti-IgE stimulation. Incubation of the cells for 24 h with 10(-6) M dexamethasone, a treatment that inhibits mediator release from human basophils, had no effect on the release of these mediators from mast cells. Dexamethasone treatment of human lung fragments led to little or no inhibition of anti-IgE-induced release of the mast cell-derived mediator, histamine, but produced a significant inhibition of the release of PGE2, PGF2 alpha, and 6-keto-PGF1 alpha. As was the case with purified mast cells, the steroid did not inhibit the release of PGD2 or TxB2 from human lung fragments. Comparison of the quantities of PGD2 and TxB2 produced by purified cells and human lung fragments reveals that the mast cells produce quantities of these metabolites sufficient to account for the entire amount produced by challenged lung fragments. Dexamethasone inhibited spontaneous release from lung fragments of all cyclooxygenase products measured. These results suggest that the human lung parenchymal mast cell phospholipase is not inhibited by dexamethasone, whereas other phospholipase(s) in the lung are inhibited by the steroid. These results may be useful in explaining the resistance of acute allergic reactions, including anaphylaxis, to steroids, despite the potent antiinflammatory activity of steroids on subacute and chronic inflammation, such as in bronchial asthma, which may be initiated by IgE-dependent mechanisms.

The characteristics of inhibition of histamine release from human lung fragments by sodium cromoglycate, salbutamol and chlorpromazine.

1 Three drugs have been tested for activity against antigen-induced histamine release from passively sensitized human lung fragments after increasing periods of pre-incubation before challenge.2 After 30 s pre-incubation, sodium cromoglycate inhibited histamine release in the concentration range 0.2-200 muM, producing a maximum inhibition of 33.0%. As the pretreatment period was extended, tolerance developed in a dose-related manner, resulting in a 48.3% and 82.8% loss of activity of the 200 muM dose after 60 min and 19 h pre-incubation, respectively. Tolerance was independent of extracellular calcium and was poorly reversible. Lung tissue desensitized to cromoglycate was cross-tolerant to the related drug, bufrolin, but not to salbutamol or chlorpromazine.3 In acute studies, salbutamol (0.03-3.0 muM) produced dose-related inhibition of histamine release, with a maximum inhibition of 72.2%. The effect was blocked stereoselectively by 1 muM propranolol, suggesting that it occurred through an interaction with lung beta-adrenoceptors. Increasing the pre-incubation time with salbutamol from 30 s to 19 h did not produce tolerance. Inhibition produced by incubation with salbutamol for 19 h was totally prevented when propranolol was added at the beginning of the pre-incubation period, indicating that it resulted from stimulation of beta-receptors and not from a non-specific or toxic effect. However, studies of reversibility of effect through washing or late addition of propranolol did indicate some change in the nature of salbutamol inhibition with time.4 Chlorpromazine was a weak inhibitor of immunological histamine release. A 100 muM concentration was ineffective after 30 s pre-incubation but its activity increased with time. Pre-incubation of lung fragments with this concentration for 1 h or longer, or with a 1 mM dose for a shorter period, provoked histamine release in the absence of antigen. Effects of chlorpromazine were not reversed by washing.5 The different characteristics shown by sodium cromoglycate, salbutamol and chlorpromazine indicate that these drugs inhibit histamine release by interfering with the secretory mechanisms in different ways.

Passive anaphylaxis in human lung fragments as a model for testing anti-allergic drugs: its variability and constraints.

Histamine release from human lung fragments, passively sensitized and challenged with antigen under standardized experimental conditions, varied between 0 and 41.2% (mean +/- SD 15.6 +/- 10.0%) in 89 experiments. Over 41 lungs, the mean coefficient of variation for release from 10 to 28 tissue replicates was 21.4% (range 7-44%). Sodium cromoglycate and chlorpromazine were both partial antagonists of histamine release producing, at best, 30-40% inhibition. The cromoglycate analogue, bufrolin, showed similar activity. There was considerable variation in the effects of these drugs both within and between experiments. Salbutamol was a more potent and more effective inhibitor of release but it, too, showed variability. Although theoretically a very appropriate model of allergic asthma, passive anaphylaxis in human lung fragments is quantitatively inconsistent and gives only a gross indication of drug efficacy.

229 Release of high molecular weight neutrophil chemotactic activity (NCA) from immunologically challenged human lung fragments

229 Release of high molecular weight neutrophil chemotactic activity (NCA) from immunologically challenged human lung fragments

Histamine-releasing properties of hydroxy-9-methyl-2-ellipticinium acetate.

Previous work on Hydroxy-9-methyl-2-ellipticinium acetate indicated a bronchoconstrictor activity which could be partially offset by antagonists of the H1 histamine receptors, and the absence of any direct effect on smooth muscle. OH-9-CH3-2-E at concentrations of 10 micrograms/ml and 500 micrograms/ml produced a moderate and a variable release of histamine when placed in contract with whole human blood and lung fragments, respectively. In addition, at a dose of 3 mg/kg in the guinea pig, pulmonary airway resistance was raised and the blood histamine level lowered. A significant correlation was found between these two effects. These results demonstrate that OH-9-CH3-2-E possesses a histamine-releasing potency which is partly responsible for its bronchial effects, implying that precautions may have to be taken when it is used as a therapeutic agent in sensitive subjects. However, the moderate intensity of this potency has not so far precluded therapeutic use of the preparation.

The role of calcium in the generation of intracellular SRS-A in passively sensitized human lung challenged with antigen.

Calcium deprivation inhibits the formation and subsequent release of SRS-A as well as histamine from passively sensitized human lung fragments challenged with antigen. Antimycin A inhibits the release of histamine and SRS-A in a dose dependant fashion, while causing a mild inhibition of SRS-A formation throughout the dose range studied. These data suggest that the influx of calcium is the critical event for the generation of SRS-A within the cell.

Inhibition of histamine release from human lung in vitro by antihistamines and related drugs.

1 A series of cationic, lipophilic histamine H1-receptor antagonists, neuroleptics, antidepressants and monoamine oxidase inhibitors were tested for their effects on anti-IgE-induced histamine release from human lung fragments in vitro. 2 They had a biphasic effect: at low concentrations a dose-related inhibition of histamine release was observed whereas, at higher concentrations, the drugs liberated histamine even in the absence of antigen. 3 Mepyramine, a much less lipophilic drug than the others tested, was only weakly active on mast cells at pharmacological concentrations. 4 The potency of the drugs as release inhibitors was not related quantitatively to their histamine liberating potency. 5 There was no; correlation between activity on mast cells and histamine H1-receptor antagonism. 6 Mast cell stabilization may play a part in the activity of these drugs as anti-allergic agents.

Secretion in dissociated human pulmonary mast cells. Evidence for solubilization of granule contents before discharge.

Mast cells were enzymatically dissociated from human lung fragments that had been sensitized with serum from human allergic to ragweed and were enriched by isopyknic and velocity gradient sedimentation. Electron microscope examination showed that the mast cells were well preserved at the end of the dissociation and isolation and that the majority of their secretory granules contained crystalline structures. These structures exhibited three patterns--scrolls, gratings, and lattices--which all could be found in the same granule. The period of crystalline structures was found to be bimodal, with maxima at 150 and 75 A. Both periods were observed in gratings that had been tilted and in scrolls that had been cut obliquely, indicating that the various gross patterns are composed of the same basic substructure. After the mast cells were stimulated by rabbit anti-human IgE to release histamine, the contents of the granule were transformed from a crystalline to an amorphous state, and only granules with amorphous contents were seen discharging from the cell. Clusters of intermediate filaments were present around the granules with amorphous contents, both deep in the cytoplasm and discharging at the cell surface. Discharge occurred both by fusion of granule membranes with the plasma membrane and by fusion of granule membranes with other granule membranes that ultimately were continuous with the plasma membrane. After discharge, the granule residue was fibrillar. Cells challenged with anti-human IgE in calcium-free medium neither released histamine nor demonstrated morphologic changes in their granules. We conclude that the crystalline state represents a storage form for materials that are solubilized before fusion of the granule membrane with the plasma membrane and discharge.

Oxatomide: inhibition and stimulation of histamine release from human lung and leucocytes in vitro.

Oxatomide is a very potent inhibitor of histamine release induced by anti-1gE and Timothy pollen extract in passively sensitized human lung fragments and that induced by anti-1gE from human leucocytes. Its spectrum of activity is different from sodium cromoglycate-like drugs. In high concentrations oxatomide, like other antihistamines and related structures, induced histamine release from both lung and leucocytes. However, oxatomide induced histamine release far more effectively from sensitized lung than non-sensitized lung. Inhibition of immunologically induced histamine releast by oxatomide may play a part in its action as an anti-hay fever and, possibly, an anti-asthmatic drug.

Histamine-containing cells from bronchial lavage of macaque monkeys. Time course and inhibition of anaphylactic histamine release.

Bronchial lavage of rhesus and cynomolgus monkeys provided leucocyte suspensions with viable histamine-containing cells (HCC) as 1--8% of the white cell population. These HCC released histamine or challenge with antiserum to human IgE. HCC from 2 monkeys with pulmonary and cutaneous hypersensitivity to Ascaris antigen (AA) released histamine on challenge with AA. The extent of histamine release was related to the concentration of antigen and antiserum, and histamine release was complete within 10 min of challenge. (+/-)Salbutamol and (-)isoprenaline were potent inhibitors of anaphylactic histamine release from HCC, but disodium cromoglycate and AH 9679 were relatively poor inhibitors. The HCC system combines the reproducibility of a cell suspension with a response to drugs similar to that of human lung fragments.

Isolation and characterization of heparin from human lung.

Heparin as measured by azure A metachromasia and anticoagulant activity has been extracted with 1 M NaCl from (35)S-labeled human lung fragments or dispersed human lung cells enriched for mast cells. The (35)S-labeled metachromatic material in the 3 M NaCl eluate from Dowex-1 chromatography of the extract from lung fragments exhibited an average mol wt of 20,000 by Sepharose 4B gel filtration. The (35)S-labeled metachromatic material with the charge characteristics of commercial porcine heparin on DEAE cellulose chromatography was entirely heparin by the criteria of resistance to degradation by chondroitin ABC lyase and complete degradation by purified heparinase. Antithrombin affinity chromatography of purified heparin with an anticoagulant activity of 137 U/mg, revealed that the one-third that was bound and eluted had a 273 U/mg sp act, whereas the unbound activity was 31 U/mg. Thus, the previously observed heterogeneity of commercial porcine heparin for binding to human antithrombin was also observed with human heparin. The mast cell-enriched human lung cell preparations yielded [(35)S]mucopolysaccharides with an average mol wt of 60,000 by Sepharose 4B gel filtration. Approximately 30% of this fraction was degraded by chondroitin ABC lyase, and the residual 70% was degraded by purified heparinase. When the chondroitin ABC lyase-resistant fraction was subjected to alkali degradation the average mol wt was reduced to 20,000. The calculated human lung mast cell heparin content of 2.4-7.8 mug/10(6) cells gave a ratio to histamine on a weight basis similar to that of intact lung fragments, thereby implying that heparin in the lung fragments was largely restricted to the mast cells.

Prostaglandin release by slow reacting substance from guinea pig and human lung tissue

Slow reacting substance (SRS) injected into the pulmonary artery released prostaglandin E (PGE) and F 2α (PGF 2α) and the 15-keto-13, 14-dihydro PG metabolites from non-sensitized and ovalbumin sensitized, isolated, perfused guinea pig lungs. PGs were also released from lungs incubated with SRS. Sensitized lungs released more PGs in both types of preparations. Indomethacin inhibited the effect of SRS. Passively sensitized human lung fragments, in parallel to guinea pig lung, released PGE, PGF 2α and the metabolites when incubatted with SRS or antigen. In in vivo experiments, SRS and arachidonic acid given intravenously increased the airway insufflation pressure in anesthetized guinea pigs. These effects, but not the action of injected PGF 2α and histamine, were abolished by indomethacin. The results indicate that one of the modes of SRS action is by release of PGs, and are consistent with the hypothesis that PGs are predominantly “secondary” mediators (in the temporal sense) of the antigen-antibody reaction.

Release of Chemical Mediators from Partially Purified Human Lung Mast Cells

Human lung mast cells dispersed by enzymatic digestion of human lung fragments were concentrated to greater than 50% purity by sedimentation in isopycnic and velocity gradients. The dispersed lung mast cells had a characteristic ultrasturctural appearance including granules with a scroll or reticular structural appearance including granules with a scroll or reticular structure surrounded by perigranular membranes. Histamine and preformed eosinophilotactic activity sedimented with mast cells on isopycnic gradients, and mast cells and these mediators were separated from the bulk of the other lung cells after velocity gradient sedimentation. The histamine content of isolated lung mast cells was calculated to range from 1.0 to 5.5 pg/cell. The quantity of SRS-A generated with anti-IgE or specific antigen was relatively limited but confined to the mast cell-rich fractions and associated with release of histamine and eosinophilotactic activity.

Allergic sensitisation of human lung fragments prevented by saturation of IgE binding sites.

PASSIVELY sensitised chopped human lung fragments release mediators of immediate hypersensitivity when challenged with appropriate antigens1. Antigen triggers the release of mediators from mast cells or basophils by coupling of neighbouring surface IgE antibody molecules sharing the same antigen specificity2. Release of mediators should therefore be unlikely if specific IgE molecules are sparsely distributed on cell surfaces, as in normal non-allergic people, or if surface receptors are densely occupied by IgE molecules with other specificities. Humans harbouring certain parasites develop high IgE levels3, and epidemiological evidence suggests that allergic disorders such as hayfever or pollen asthma are rare in heavily parasitised populations4,5. It is not known whether IgE induced in man by parasite infestation is directed specifically against parasite antigens, or whether it represents nonspecific potentiation of IgE production against many different antigens, as demonstrated in the rat Nippostrongylus model6. Here we show that human lung fragments first exposed to IgE-rich serum from West African residents become resistant to further passive sensitisation with serum containing grass pollen-specific IgE.

Purification and synthesis of eosinophilotactic tetrapeptides of human lung tissue: identification as eosinophil chemotactic factor of anaphylaxis.

Preferential eosinophil chemotactic activity exhibiting a molecular weight comparable to that released from sensitized human lung fragments challenged with specific antigen and designated eosinophil chemotactic factor of anaphylaxis has been isolated from extracts of human lung fragments by sequential purification on Sephadex G-25, Dowex-1, Sephadex G-10, and paper chromatography. Two eosinophilotactic tetrapeptides of amino acid sequence Val-Gly-Ser-Glu and Ala-Gly-Ser-Glu were recovered from the extracts in 4-12% overall yield of the low molecular weight peak from Sephadex G-25. Purified eosinophil chemotactic factor of anaphylaxis and the synthetic tetrapeptides were maximally active in amounts from 0.1 to 1.0 nmol per chemotactic chamber, and the activity was dependent on both the NH2-terminal and the COOH-terminal residues. Both natural and synthetic peptides were preferentially chemotactic for eosinophils and rendered them unresponsive to a subsequent stimulus.