Inactivation by alveolar macrophages of slow-reacting substance of anaphylexis released from pig lung cells

The influence of alveolar macrophages on the release of slow-reacting substance of anaphylaxis (SRS-A) and histamine from dispersed pig lung cells was studied. The addition of alveolar macrophages to lung cells (1:3) decreased SRS-A release by 62% ± 7% without a significant decrease in histamine release. Increased numbers of alveolar macrophages (1:1) decreased both SRS-A release (by 90% ± 5%) and to a lesser extent histamine release (by 59% ± 18%). The decrease in released SRS-A was not caused by prostaglandin release from macrophages, and kinetic data indicated that in contrast to histamine the decrease was predominantly the result of SRS-A inactivation rather than inhibition of its release. When alveolar macrophages were incubated with SRS-A, inactivation (191 units /10⁷ cells/30 min) was confirmed. SRS inactivation was not reproduced by glutaraldehyde-treated macrophages or by granulocyte and mononuclear populations obtained from peripheral blood. Alveolar macrophages may limit the consequences of SRS-A release in the peripheral air spaces.     

The effect of Ro 21-7634 on the antigen-induced production of bronchoconstrictive arachidonic acid metabolites in the guinea pig lung

Ro 21-7634 has previously been shown to inhibit histamine and SRS-A release from actively-sensitized guinea pig lung fragments upon antigen challenge. In the studies described herein, it was observed that Ro 21-7634 does not decrease SRS-A release but instead acts to inhibit the synthesis of this mediator. This was confirmed by studying SRS-A synthesisin vitro in rat peritoneal cells after challenge with ionophore A23187. In the peritoneal cell system, Ro 21-7634 exhibited an IC₅₀ of 500 M, in comparison with 5,8,11,14-eicosatetraynoic acid, phenidone and BW755C (IC₅₀'s of 2, 100, and 100 M, respectively). When studied at 10^–4 and 10^–3 M in perfused guinea pig lung, Ro 21-7634 inhibited antigen-induced thromboxane A₂ production by 68 and 96%, respectively. In this system, antigen is believed to induce thromboxane A₂ production through the release of histamine and SRS-A from lung tissue. These mediators then interact at receptor sites in the lung parenchyma to induce thromboxane A₂ synthesis. Ro 21-7634 could thus be inhibiting thromboxane A₂ production by preventing the release of histamine and synthesis of SRS-A in the perfused lung system. Such a mechanism is suggested by the fact that although Ro 21-7634 was effective in inhibiting antigen-induced thromboxane production, it was ineffective in inhibiting thromboxane A₂ production induced in the guinea pig lung system by the direct perfusion of histamine or SRS-A through the lung.     

Role of Phospholipase A2 Activation in Histamine Release from Human Basophils

The present experiments were undertaken to investigate the role of phospholipase A₂ (PLA₂) activation in histamine release from human basophils. A PLA₂ inhibitor, P-bromophenacyl bromide (BPB), inhibited IgE-mediated anti-IgE-induced histamine release from human basophils with a concentration of drug required to produce 50% inhibition (IC₅₀) of 1.5 × 10^?6 m when leukocytes were preincubated with this agent for 15 min. Histamine release induced by calcium ionophore A23187 and formyl-l-methionyl-l-leucyl-l-phenylalanine was also blocked by BPB with IC₅₀ of 4.1 × 10^?6 m, and 3.5 × 10^?6 m, respectively. A PLA₂ activator, 12–0-tetradecanoylphorbol-13-acetate (TPA) caused basophil histamine release with a dose-dependent fashion. BPB inhibited TPA-induced histamine release (IC₅₀: 2.5 × 10^?6 m). However, another PLA₂ activator, melittin, and PLA₂ did not release histamine through non-cytotoxic mechanisms. Collectively, these results suggest that PLA₂ activation plays a central role in histamine release from human basophils via generation of lysophosphatidylcholine or products of the lipoxygenase pathway of arachidonic acid metabolism.     

Effect of arachidonic acid metabolism on the release of histamine and SRS (leukotrienes) from guinea-pig lung

The effect of arachidonic acid (AA) metabolism on histamine release and SRS (leukotrienes) production has been studied in guinea-pig lung using anaphylactic reaction and Ca²⁺ ionophore as the triggering agentsin vitro. AA andl-cysteine enhanced SRS production without any appreciable effect on histamine release. Two nonsteroid anti-inflammatory agents, indomethacin and ketoprofen, which block prostaglandin production by the cyclooxygenase pathway, stimulated SRS production but had hardly any effect on histamine release, indicating that SRS synthesis is more sensitive to prostaglandin regulation. Enhancement of SRS production was more pronounced for antigen than for Ca²⁺ ionophore. This might be related to different cellular origin of SRS with the two triggering agents. Using rat perltoneal cells, both mast cells and the other cells were found to produce SRS in response to Ca²⁺ ionophore, the amount formed by the latter type of cells being higher. Inhibition of lipoxygenase by 5,8,11,14-eicosatetraynoic acid and nordihydroguaiaretic acid depressed SRS production, but had no effect on histamine release. SRS production triggered by Ca²⁺ ionophore was more sensitive, possibly because of different cellular origin of SRS in response to the two stimuli. The explanation for the discrepancy between the effect on SRS production and histamine release may also have to be sought in their different origins. SRS may mainly stem from cells, which are more sensitive to the inhibitors than the mast cell, which is the source of histamine.     

An in vitro model of canine immediate-type hypersensitivity reactions.

Fragmented lung prepared from dogs cutaneously sensitive to ascaris antigen released histamine and a slow-reacting substance of anaphylaxis-like (SRS-A) material upon antigen challenge. Passive sensitization of fragments with serum obtained from ascaris-sensitive donor dogs enhanced the release of both substances; heating (56 degrees C for 4 h) destroyed the ability of the serum to enhance release. In passively sensitized tissues isoproterenol and papaverine inhibited the release of histamine and SRS-A; propranolol antagonized the effect of isoproterenol. Carbachol enhanced the release of both substances from passively sensitized lung wheras disodium cromoglycate and aminoguanidine were without effect. It is concluded that fragmented canine lung is a useful model for study of immediate-type hypersensitivity reactions.     

Pharmacologic regulation of antigen-induced mediator release from canine lung.

The ascaris antigen-induced release of histamine and a slow-reacting substance of anaphylaxis (SRS-A) from passively sensitized fragmented canine lung is further characterized. Histamine and SRS-A were released within 30 sec of antigen challenge, reached a maximum at 7 and 10 min, respectively, and thereafter appeared to remain constant to 30 min. Contractions of guinea pig ileum produced by canine SRS-A were competitively antagonized by the SRS-A antagonist FPL-55712. Indomethacin and deuterium oxide enhanced antigen-induced SRS-A release from canine lung but had little effect on histamine release. The ability of several chemically novel 'antiallergic agents' to inhibit mediator release was evaluated. Inhibition of histamine release, and to a lesser extent SRS-A release, by one of these compounds was shown to vary with time and temperature. It is concluded that fragmented canine lung, while disclosing some qualitative pharmacological differences from other species, is a useful in vitro model of immediate hypersensitivity reactions.     

In vitro studies on the mechanism of action of a new antiallergic,Ro 21-7634

The effects of Ro 21-7634 and disodium cromoglycate (cromoglycate) on the in vitro release of mediators of anaphylaxis from rat peritoneal cells and guinea pig lung tissue were compared. Ro 21-7634 was 25 fold more potent than cromoglycate as an inhibitor of antigen-induced histamine release from passively sensitized (IgE) rat peritoneal cells. Ro 21-7634 was also the more potent inhibitor of both compound 48/80- and concanavalin A-induced histamine release from rat peritoneal cells. The two drugs shared the common properties of producing the same maximal level of inhibition in each of the above releasing systems and exhibiting a time and concentration dependent loss of inhibitory activity when added to the cells prior to the releasing agent. Neither drug inhibited ionophore A23187- or ionophore X537A-induced histamine release from these cells. Ro 21-7634 inhibited antigen-induced (IgG₁) histamine and SRS-A release from actively sensitized guinea pig lung fragments, whereas cromoglycate did not. The results indicate that Ro 21-7634 and cromoglycate act through a common mechanism to inhibit allergic mediator release and that Ro 21-7634 is the more potent inhibitor.     

Histamine release from serosal mast cells by intermediate products of arachidonic acid metabolism

In the present paper we report the results of experiments carried out to measure the release of histamine from isolated rat mast cells during the metabolic activation of arachidonic acid. Arachidonic acid (10^–8–10^–4 M) and the terminal products (10^–6 M) of the arachidonic acid pathways were devoid of any significant histamine releasing properties. A substantial amount of histamine was released from rat mast cells by low concentrations of arachidonic acid during incubation with prostanoid generating systems, such as guinea-pig lung microsomes, rat serosal macrophages and polymorphonuclear cells and prostaglandin-H-synthase from calf seminal vesicles. The release of histamine was not accompanied by a leakage of lactate dehydrogenase and was blocked byd-mannitol and by lipoxygenase and cyclo-oxygenase pathway inhibitors. The data are consistent with the hypothesis that free radical derivatives of arachidonic acid, originating from hydroperoxy fatty acids, are generated during catalysis, causing mast cell histamine release.     

Inhibition of histamine release from human lung and rat peritoneal mast cells by cyclosporin-A

Cyclosporin A (CS-A) partly inhibited IgE-mediated histamine release from human lung tissuein vitro (chopped and collagenase-dispersed preparations). Inhibition started at concentrations within the clinical blood level of the drug, but the IC₅₀ was much higher (10–50 M; 50% inhibition reached only in some experiments). CS-A also inhibited histamine release from rat peritoneal mast cells (RPMC) induced by antigen, concanavalin-A (Con-A), compound 48/80 and ionophore A23187. The IC₅₀ values were 0.3, 23.0, and 33.0 M for Con-A, A23187 and ovalbumin respectively. Inhibition of 48/80-induced release did not reach 50%. By comparison with human basophils the human lung and RPMC were less sensitive to the inhibitory action of CS-A. The IgE-mediated Schultz-Dale reaction in human lung strips was slightly and inconsistently inhibited by CS-A, but IgG₁-mediated reaction in guinea-pig lung strips was potentiated by the drug.     

Differentiation of second messenger systems in mast cell activation

Pretreatment of rat peritoneal mast cells with either Staurosporine or an analog K-252a, lead to a dose-related inhibition of histamine release when stimulated with Anti-IgE (IC₅₀: Staurosporine=110 nM; K-252a=100 nM). In contrast, the two PKC inhibitors (1–1000 nM) failed to inhibit histamine release induced by compound 48/80 (0.5–1 g/ml). Exposure of Anti-Asc-IgE sensitized mouse bone marrow derived mast cells to Asc-BSA lead to the release of both histamine (510 ng±12.6 ng/10⁶ cells) and immunoreactive Leukotriene C₄ (27.0±12.6 ng/10⁶ cells) LTC₄ release was inhibited by Staurosporine and K-252a with an IC₅₀ of 75 nM for both compounds. Pretreatment of rat peritoneal mast cells with PMA 100 nM lead to a small but significant release of histamine (18.3±3.6%). Pretreatment of these cells with K-252a or Staurosporine lead to a dose related inhibition of histamine release with an ED₅₀ of 10 nM for Staurosporine and 60 nM for K-252a. Treatment of rat peritoneal mast cells with the calcium ionophore A23187 lead to a significant release of histamine which was not inhibited by either of the two kinase inhibitors (0.1–1000 nM). The two kinase inhibitors also inhibited mouse bone marrow derived mast cell proliferation in response to IL-3 with IC₅₀ of 80 nM for Staurosporine and 270 nM for K-252a.     

Release of slow-reacting substance from anaphylactic lung tissue and its modification by beta-sympathomimetics.

Anaphylactic release of slow-reacting substance of anaphylaxis (SRS-A) from chopped guinea pig lung was studied. The antibodies mediating the anaphylactic reaction were classified as IgG antibodies by PCA technique. Isoprenaline was found to inhibit the release of SRS-A in the concentration range 10-8-10-6M, while the beta2-selective agonist terbutaline showed inhibition in the concentration range 10-6-10-5M. The beta1-selective beta-agonist tazolol was ineffective when tested in the concentration range 10-7-10-5M. The rank order of inhibitory potency of the compounds on SRS-A release agree with that for histamine release.     

Passive sensitization of guinea-pig skin in vitro for the antigen-induced release of anaphylactic mediators

Guinea-pig skin fragments were passively sensitized for the antigen-induced release of histamine, SRS-A and ECF-A. Skin histamine was chemically identified by fluorimetry; SRS-A gave a characteristic dose-dependent contraction of the guinea-pig ileum; and ECF-A selectively attracted eosinophils. The antibody mediating the release of these agents was shown to be IgG₁. Following antigen challenge different time courses of release were demonstrable for histamine, SRS-A and ECF-A. Skin SRS-A was resistant to treatment with the enzyme pronase and skin ECF-A had an estimated molecular size of between 500 and 1000. They were therefore comparable to similar agents released from the lung. Thus SRS-A and ECF-A join histamine as chemical mediators in cutaneous anaphylaxis.     

The effect of a new antihistamine drug,Loderix® (EGIS-2062), on stimulus-evoked histamine release from rat peritoneal mast cells

The ability of a new, non-sedative antihistamine drug, Loderix^® (EGIS-2062), to inhibit stimulusevoked histamine release from rat peritoneal mast cells has been investigated and compared with that of ketotifen (Zaditen^®). At low concentrations Loderix^® preincubated with the cells for 10 min prior to the addition of various stimulants (immune aggregates, ionophore A23187 and compound 48/80) produced a concentration-dependent inhibition of histamine release, while at high concentrations it induced the release of histamine. The IC₅₀ values were calculated as 0.2, 15 and 50 M by using immune aggregate, (rat IgG₂+anti rat IgG) calcium ionophore and 48/80 as stimulants, respectively. At the IC₅₀ level Loderix^® was more effective than Zaditen^® (100 times more effective when immune aggregate and 4 times when 48/80 were used for stimulation). Both drugs had a dual effect on mast cells. The morphological observations confirmed the pharmacological action of the drugs, showing also that the histamine release at high concentrations is due to their cytotoxic effect.     

Peritoneal anaphylaxis in the rat after sensitization with mouse antiserum.

Passive peritoneal anaphylaxis in rats, sensitized with mouse antiserum, had characteristics of an IgE-mediated reaction, in that the serum was heat-labile and pretreatment of the rats with disodium cromoglycate (DSCG), or sodium nivimedone, inhibited the release of both histamine and slow-reacting substance of anaphylaxis (SRS-A). Sodium nivimedone was more potent than DSCG as an inhibitor of histamine release. Peak concentrations of histamine and SRS-A in the peritoneal fluids of the rats, were reached within 2 min of antigen challenge and fell to control levels after 20-30 min.     

Mechanism of the action of amoxanox (AA-673), an orally active antiallergic agent

Amoxanox inhibited immunologically stimulated and LTD4-induced bronchoconstriction in laboratory animals. Amoxanox, like DSCG, inhibited rat IgE-mediated PCA and histamine release from rat peritoneal mast cells, and suppressed immunologically stimulated or calcium ionophore A23187-induced SRS-A generation in rat peritoneal cavity and guinea pig lung fragments. This compound also reduced the contractile response of guinea pig lung parenchymal and ileal strips to LTD4, but did not significantly affect the response of the ileum to either histamine or acetylcholine. Therefore, the antiallergic action of amoxanox seems to be associated with inhibition of chemical mediator release and antagonistic activity on SRS-A.     

Modulation of the release of SRS-A from bovine lung in vitro by several autonomic and autacoid agents.

The immunological release of slow-reacting substance of anaphylaxis (SRS-A) from bovine lung is modulated by several autonomic and autacoid agents. Isoproterenol, a beta-adrenergic agonist, inhibited release of SRS-A, as did cyclic AMP. Phenylephrine also inhibited SRS-A release but through alpha-adrenergic mechanisms. This is in contrast to observations in human lung. Carbachol enhanced the release of bovine SRS-A. Histamine inhibited SRS-A release through the H2-receptor. Dopamine enhanced the release of SRS-A by means of a dopaminergic receptor. 5-HT did not significantly modulate SRS-A release.     

The Orally Active Antiallergic Compound,LC-6 (Trans-2,3b,4,5,7,8b,9,10-Octahydronaphtho[l,2-C:5,6-C] Dipyrazole) Inhibits the Arachidonate Lipoxygenase Enzyme

Abstract

Trans-2,3,4,5,7,8b,9,10-octahydronaphtho[l,2-C:5,6-C]dipyrazole (LC-6), which is active in vivo when administered orally, inhibits histamine release from peritoneal rat mast cells and human basophils in vitro. LC-6.2C1 blocks both IgE-mediated and non-IgE-mediated histamine release. Histamine release induced by antigen, dextran, compound 48/80, or the Ca²⁺ ionophore A23187 was effectively inhibited. The in vitro data indicate that LC6. 2HC1 has the capacity to inhibit the mediator release phase of anaphylaxis. The drug has the ability to inhibit arachidonate lipoxygenase activity. The IC50 for IgE-mediated histamine release inhibition is in good agreement with the IC50 for inhibition of arachidonate lipoxygenase activity. The data indicate that LC-6 acts in vivo by blocking the release of mediators; its activity may be explained by its ability to stop the synthesis of lipoxygenase-derived arachidonic acid products.     

Oxatomide inhibits the release of bronchoconstrictor arachidonic acid metabolites (iLTC4 and PGD2) from rat mast cells and guinea-pig lung

The effect of oxatomide, an orally active antiallergic drug, on immunoreactive LTC₄ (iLTC₄) production has been studied in rat peritoneal exudate cells (PEC) and guinea-pig lung fragments using the calcium ionophore A23187 and specific antigen in vitro. Oxatomide (10^–5 M) inhibited iLTC₄ release by 70% with A23187 from rat PEC, and by 48% with antigen from guinea-pig lung. Oxatomide is supposed to affect the biosynthesis pathway of leukotrienes, because oxatomide inhibits 5-lipoxygenase from guinea-pig peritoneal leukocytes with an IC₅₀ 17 M. Oxatomide also depressed the release of PGD₂ from rat peritoneal mast cells stimulated by A23187 (IC₅₀ 4.2 M). The effects of oxatomide on iLTC₄ and PGD₂ release were more potent than other antiallergic drugs (DSCG, ketotifen, tranilast).     

Effects of leukotrienes C4 and D4 on guinea-pig heart and the participation of SRS-A in the manifestations of guinea-pig cardiac anaphylaxis

Synthetic leukotrienes C₄ and D₄ (LTC and LTD) were found to possess potent coronary vasoconstrictor and cardiac depressant actions on isolated guinea-pig hearts. We therefore went further to investigate the possibility that endogenously released slow-reacting substance of anaphylaxis (SRS-A) might be responsible for the coronary vasoconstriction and negative inotropism in guinea-pig cardiac anaphylaxis. Results using time-course analysis as well as the specific SRS antagonist FPL 55712 have shown that SRS-A released during cardiac anaphylaxis was unlikely to be responsible for the early and most dramatic phase of coronary vasoconstriction that usually occurred at the 2nd min after antigen challenge, but could possibly be responsible for the latter and more prolonged phase occurring between the 6th and 14th min. This is because SRS-A release was found to peak at the 4th min after antigen challenge, 2 min after vasoconstriction had already peaked. Moreover, this early component of coronary vasoconstriction could not be blocked by FPL 55712, whereas the latter component was significantly reduced by the antagonist. The negative inotropism following cardiac anaphylaxis was also found to be significantly reduced by FPL 55712, thus suggesting SRS-A involvement. However, our experiments did not show whether the two actions were direct effects of SRS-A or whether contractility failure was a consequence of coronary vasoconstriction.     

Granulocyte-macrophage colony-stimulating factor enhances basophil histamine release induced by non-IgE-dependent stimulators

Some cytokines are known to affect IgE-mediated basophil histamine release. The effect of the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) on human basophil and masct cell histamine release was studies further. Blood leukocytes with approximately 2% basophils from 9 healthy individuals were incubated with recombinant human GM-CSF (0.3–30 U/ml) in combination with A23187 (10^–7–10^–6 M) or washedStaphylococcus aureus whole bacteria (0.3–2.5 mg/ml). Histamine release was measured spectrofluorometrically. GM-CSF in itself did not induce histamine release. The addition of GM-CSF to cells stimulated with A 23187 caused a dose-dependent enhancement amounting to mean 70% at 3 U/ml and mean 170% at 30 U/ml (P<0.05). GM-CSF enhanced the bacteria-induced histamine release by 30% at U/ml and by 65% at 3 U/ml (P<0.05). The enhancement did not depend on cell-bound IgE or LPS contamination. In preliminary mast cell experiments with lung tissue we did not find an enancing effect of GM-CSF on IgE-mediated histamine release.