Antigen-induced Mediator Release Research Articles

High affinity IgE-Fc receptor α and γ subunit interactions.

To explore the relationships between the subunits (α, β and γ) of the high affinity IgE receptor (Fc&RI) and its ability to mediate transmembrane signaling. Experimental study. Department of Molecular Biology and Biotechnology, University of Sheffield, UK, from 2008 to 2009. The approach employed was to create a chimera (human αγγ) using the extracellular (EC) domain of the human high affinity IgE receptor. The alpha subunit (huFc&RIα) of IgE receptor was spliced onto the rodent gamma TM and cytoplasmic domain (CD). This was transfected into the Rat Basophilic Leukemia cell line in order to assess the possibility of selectively activating cells transfected with this single pass construct for antigen induced mediator release. The RBLs cell lines transfected with the huFc&RIα/γ/γ cDNA constructs were assessed for the cell surface expression of the huFc&RIα subunit and the response to the antigenic stimulus by looking for degranulation and intracellular Ca2+ mobilisation. The results obtained showed the absence of huFc&RIα subunit expression on the surface of transfected cells as seen by flowcytometric studies, β-hexosaminidase assays and intracellular calcium mobilisation studies. In the present study the grounds for non-expression of huFc&RIα/γ/γ cDNA remains elusive but may be due to the fact that the human-rodent chimeric receptors are assembled differently than the endogenous rodent receptors as seen in study in which COS 7 cells were transfected with human/rat chimeric complexes.

Effects of 7,8‐dihydro‐8‐oxo‐deoxyguanosine on antigen challenge in ovalbumin‐sensitized mice may be mediated by suppression of Rac

Earlier we reported that 7,8-dihydro-8-oxo-deoxyguanosine (8-oxo-dG), an oxidatively modified guanine nucleoside, exerted anti-inflammatory activity through inactivation of the GTP binding protein, Rac. In the present study, the effects of 8-oxo-dG were investigated on responses to antigen challenge in sensitized mice, as Rac is also involved at several steps of the immune process including antigen-induced release of mediators from mast cells. Mice were sensitized and challenged with ovalbumin without or with oral administration of 8-oxo-dG during the challenge. Effects of 8-oxo-dG were assessed by measuring lung function, cells and cytokines in broncho-alveolar lavage fluid (BALF) and serum levels of antigen-specific IgE. Rac activity in BALF cells was also measured. 8-oxo-dG inhibited the increased airway resistance and decreased lung compliance of sensitized and challenged mice to the levels of non-sensitized control mice and lowered the increased leukocytes particularly, eosinophils, in BALF. Furthermore, 8-oxo-dG suppressed allergy-associated immune responses, such as raised anti- ovalbumin IgE antibody in serum, increased expression of CD40 and CD40 ligand in lung, increased interleukin-4, -5, -13, interferon-gamma and tumour necrosis factor-alpha in BALF and mRNA levels of these cytokines in BALF cells, dose-dependently. The corresponding purine, 8-oxo-guanine, showed no effects in the same experiments. Finally, 8-oxo-dG, but not 8-oxo-guanine, inhibited the increased Rac activity in sensitized and challenged mice. 8-Oxo-dG had anti-allergic actions that might be mediated by Rac inactivation. This compound merits further evaluation of its therapeutic potential in allergic asthma.

Nitric oxide positively regulates Ag (I)-induced Ca2+ influx and mast cell activation: role of a nitric oxide synthase-independent pathway

NO is generated by NOS activity and known to act as a negative regulator of mast cell activation. We reported previously that Ag (I) directly evokes mast cell degranulation and LTC(4) release via Ca(2+) influx through thiol-sensitive, store-independent channels. Here, we report that NO generated independently of NOS activity mediates the store-independent Ca(2+) influx. Exposure of mast cells to Ag (I) resulted in increased intracellular NO levels and NO(2)(-)/NO(3)(-) contents in the extracellular fluid. The NO increase was blocked by NO scavenger Hb and DTT but not by NOS inhibitors such as amino-BH(4) and L-NAME. This NO production occurred independently of the Src family kinase and PI3K activities, both of which were necessary for antigen-induced, NOS-dependent NO production. Hb and DTT reduced Ag (I)-induced beta-hexosaminidase release and LTC(4) release, whereas the NO scavengers and NOS inhibitors augmented antigen-induced mediator release. Moreover, Hb and DTT, but not the NOS inhibitors, abolished the Ag (I)-induced Ca(2+) influx, and none of the drugs blocked CRAC channel activity. Finally, Ag (I)-induced Ca(2+) influx was distinct from LTCC activity in terms of its sensitivities to wortmannin and LTCC antagonists and the effects of Ca(v)1.2 LTCC gene silencing. These data show that NOS-independent NO regulates mast cell activation positively via a unique store-independent Ca(2+) influx pathway. The present findings suggest multiple sources and functions of NO in mast cell biology.

Effects of selective and non-selective COX inhibitors on antigen-induced release of prostanoid mediators and bronchoconstriction in the isolated perfused and ventilated guinea pig lung

The contribution of cycloxygenase (COX)-1 and COX-2 in antigen-induced release of mediators and ensuing bronchoconstriction was investigated in the isolated perfused guinea pig lung (IPL). Antigen challenge with ovalbumin (OVA) of lungs from actively sensitised animals induced release of thromboxane (TX)A 2, prostaglandin (PG)D 2, PGF 2 α , PGI 2 and PGE 2, measured in the lung effluent as immunoreactive TXB 2, PGD 2-MOX, PGF 2 α , 6-keto PGF 1 α and PGE 2, respectively. This release was abolished by the non-selective COX inhibitor flurbiprofen (10 μM). In contrast, neither the selective COX-1 inhibitor FR122047 nor the selective COX-2 inhibitor celecoxib (10 μM each) significantly inhibited the OVA-induced bronchoconstriction or release of COX products, except for PGD 2. Another non-selecive COX inhibitor, diclofenac (10 μM) also significantly inhibited antigen-induced bronchoconstriction. The data suggest that both COX isoenzymes, COX-1 and COX-2 contribute to the immediate antigen-induced generation of prostanoids in IPL and that the COX-1 and COX-2 activities are not associated with different profiles of prostanoid end products.

Generation of canine-human Fc IgE chimeric antibodies for the determination of the canine IgE domain of interaction with FcɛRIα

Identification of the domain(s) of canine IgE that interact with FcɛRIα may lead to novel therapeutic intervention strategies that inhibit the ability of canine IgE to engage FcɛRIα. A panel of canine-human Fc IgE chimeric antibodies was constructed to investigate this interaction by replacing canine IgE-Fc domains with the corresponding human IgE-Fc domains since human IgE-Fc does not recognize canine FcɛRIα. β-Hexosaminidase release assays were performed to assess the ability of the chimeric antibodies to bind to and sensitize a novel RBL cell line transfected with canine FcɛRIα for antigen induced mediator release. Replacing canine Cɛ2 with human Cɛ2 resulted in similar levels of release as those elicited by canine Fc IgE from RBL-2H3 cells transfected with either canine FcɛRIα or human FcɛRIα. Substitution of canine Cɛ4 with human Cɛ4 resulted in approximately 10% lower levels of release compared to cells sensitized with canine Fc IgE. Receptor binding by flow cytometry and cell activation could not be detected when transfected RBL cells were incubated with chimeric constructs where canine Cɛ2 and Cɛ4 were substituted with human Cɛ2 and Cɛ4. However, when this construct was incubated with cognate antigen prior to cell challenge mediator release was observed, albeit at a 20% lower level, indicating that while canine Cɛ3 is the only domain essential for binding to canine or human FcɛRIα, species specific residues in canine Cɛ2 and Cɛ4 inhibit dissociation of the ligand from the receptor.

Environmental Polycyclic Aromatic Hydrocarbons (PAHs) Enhance Allergic Inflammation by Acting on Human Basophils

Diesel exhaust particles (DEPs) have been implicated in the worldwide increased incidence of allergic airway diseases over the past century. There is growing evidence that DEP-associated polycyclic aromatic hydrocarbons (PAHs) participate in the development and maintenance of immunoglobulin (Ig) E-mediated allergic diseases. To address this issue we investigated the impact of U.S. Environmental Protection Agency (EPA) priority PAHs as well as of PAH-containing airborne extracts on antigen-induced CD63 upregulation and mediator release from human basophils. Whole blood samples from birch pollen allergic and control subjects were incubated in the presence of organic extracts of urban aerosol (AERex) or EPA-PAH standard with or without rBet v 1. Basophils were analyzed for CD63 expression as a measure of basophil activation by using multiparameter flow cytometry. In addition, purified basophils from birch pollen allergic donors were incubated for 2 h in the presence of 1 μM benzo[a]pyrene (BaP) or phenanthrene (Phe) and then stimulated with rBet v 1 for 45 min. Supernatants were assayed for histamine, interleukin (IL)-4, and IL-8 by means of enzyme-linked immunosorbent assay (ELISA). Basophils exposed in vitro simultaneously to AERex or EPA-PAH standard and rBet v 1 expressed CD63 significantly more than with antigen alone. PAHs synergized with rBet v 1 dose dependently, but did not activate basophils from nonallergic donors. BaP and Phe significantly enhanced cytokine secretion (IL-4, IL-8) and histamine release from purified basophils without antigen added, and secretion was not further enhanced by rBet v 1 stimulation. In conclusion, PAHs from roadside emissions can directly activate sensitized basophils to cytokine secretion and drive proallergic processes through enhanced Fcε RI-coupled mediator release from human basophils.

Effect of cyclosporin and tacrolimus (FK506) on the antigen-induced mediator release, membrane potential and 86Rb +/K + and Ca 2+ fluxes in the RBL-2H3 cell line

The immunosuppressants cyclosporin A (CsA) and tacrolimus (FK506) inhibit the activation by antigen of T-lymphocytes as well as mast cells. The mechanism of their action on mast cells has yet to be elucidated. We, therefore, assessed their effect on antigen-induced histamine and β-hexosaminidase release, membrane potential changes (bis-oxonol fluorescent probe), 86RB + (marker for K +)-efflux, the intracellular free calcium concentration ([Ca 2+] i in single cells) and 45Ca 2+ uptake (CsA only) in RBL-2H3 cells, a mucosal-type mast cell line, passively sensitized with monoclonal mouse IgE antibody. Antigen addition induced depolarization within 1–2 min, followed by slower repolarization, reaching a steady state (∼90% repolarization) after 7–9 min. CsA and FK506 each dose-dependently inhibited antigen-induced histamine and β-hexosaminidase secretion and the membrane repolarization phase, with similar IC 50s for both actions, ∼20 nM for CsA and ∼2 nM for FK506. Antigen-induced 86Rb +-efflux was also significantly inhibited. Antigen-evoked increase in [Ca 2+] i (area under the curve, AUC) was reduced by 35% and 52% in the presence of CsA or FK506 (1 μM each), respectively. However, 45Ca 2+-uptake was not inhibited by CsA. These results suggest that both CsA and FK506 may inhibit mediator release from mast cells via blocking two interrelated processes, which are involved in the secretory process: 1. Membrane repolarization phase, which is essential for optimal mediator secretion and is mediated by a Ca 2+-sensitive K +-efflux, yet to be further characterized, and (2) Increase in [Ca 2+] i , probably via reduction of Ca +2-release from intracellular stores, [Ca 2+] s.

Co-aggregation of FcγRII with FcϵRI on Human Mast Cells Inhibits Antigen-induced Secretion and Involves SHIP-Grb2-Dok Complexes

Signaling through the high affinity IgE receptor FcepsilonRI on human basophils and rodent mast cells is decreased by co-aggregating these receptors to the low affinity IgG receptor FcgammaRII. We used a recently described fusion protein, GE2, which is composed of key portions of the human gamma1 and the human epsilon heavy chains, to dissect the mechanisms that lead to human mast cell and basophil inhibition through co-aggregation of FcgammaRII and FcepsilonRI. Unstimulated human mast cells derived from umbilical cord blood express the immunoreceptor tyrosine-based inhibitory motif-containing receptor FcgammaRII but not FcgammaRI or FcgammaRIII. Interaction of the mast cells with GE2 alone did not cause degranulation. Co-aggregating FcepsilonRI and FcgammaRII with GE2 1) significantly inhibited IgE-mediated histamine release, cytokine production, and Ca(2+) mobilization, 2) reduced the antigen-induced morphological changes associated with mast cell degranulation, 3) reduced the tyrosine phosphorylation of several cellular substrates, and 4) increased the tyrosine phosphorylation of the adapter protein downstream of kinase 1 (p62(dok); Dok), growth factor receptor-bound protein 2 (Grb2), and SH2 domain containing inositol 5-phosphatase (SHIP). Tyrosine phosphorylation of Dok was associated with increased binding to Grb2. Surprisingly, in non-stimulated cells, there were complexes of phosphorylated SHIP-Grb2-Dok that were lost upon IgE receptor activation but retained under conditions of Fcepsilon-Fcgamma co-aggregation. Finally, studies using mast cells from Dok-1 knock-out mice showed that IgE alone triggers degranulation supporting an inhibitory role for Dok degranulation. Our results demonstrate how human FcepsilonRI-mediated responses can be inhibited by co-aggregation with FcgammaRIIB and implicate Dok, SHIP, and Grb2 as key intermediates in regulating antigen-induced mediator release.

Inhibition of the antigen-induced activation of RBL-2H3 cells by charybdotoxin and cetiedil

Quinidine and Ba 2+, non-selective K +-channel blockers, have previously been shown to inhibit antigen-induced mediator (β-hexosaminidase) release from RBL-2H3 cells, a mucosal-type mast cell line. We therefore used selective blockers of Ca 2+-activated and other K + channels to determine if there was a role for these channels in antigen-induced mediator release. Charybdotoxin and cetiedil dose-dependently inhibited β-hexosaminidase release with IC 50 values of 133 nM and 84 μM, respectively. Charybdotoxin also inhibited the repolarization phase of the antigen-induced biphasic change in the membrane potential (IC 50 84 nM), antigen-stimulated 86Rb +-efflux and increase in free intracellular calcium, [Ca 2+] i. Iberiotoxin, margatoxin, apamin and tetraethylammonium had no effect on β-hexosaminidase release. These results suggest that K + conductances play a significant role in mediator release from RBL-2H3, that these conductances are of the intermediate conductance Ca 2+-activated K + channel (IK Ca) type, and that they are somewhat similar to those which have been described in red blood cells, though they are much less sensitive to clotrimazole.

Inhibition of antigen-induced mediator release from IgE-sensitized cells by a monoclonal anti-Fc epsilon RI alpha-chain receptor antibody: implications for the involvement of the membrane-proximal alpha-chain region in Fc epsilon RI-mediated cell activation.

The interaction between human IgE and its high affinity receptor, FcepsilonRI, is a critical event in mediating the allergic response. Aggregation of the alpha-chain of FcepsilonRI (FcepsilonRIalpha) occurs via cross-linking of receptor-bound IgE by Ag, resulting in cell activation and the release of mediators of hypersensitivity. Recently, we mapped the epitopes of two anti-FcepsilonRIalpha mAbs, 15/1 and 5H5F8. In contrast to 15/1, mAb 5H5F8 does not inhibit IgE binding to FcepsilonRIalpha. Here we demonstrate both 5H5F8 binding to FcepsilonRI(+) cells as well as a high level of IgE binding to 5H5F8-saturated cells. At the same time 5H5F8 strongly inhibits hexosaminidase release and Ca(2+) flux after Ag triggering from human IgE-sensitized RBL-2H3 cells stably transfected with human FcepsilonRIalpha. Further, 5H5F8 and its Fab inhibit sulfidoleukotriene and histamine release from primary human peripheral blood leukocytes, including cells bearing endogenous IGE: Furthermore, we confirm that 5H5F8 maps to a linear peptide sequence in close proximity to the cell membrane. Two chemically synthesized peptides containing the 5H5F8 epitope sequence PREKY were selected for detailed analysis of 5H5F8 and 5H5F8 Fab binding and were found to produce K(d) values of similar magnitude to that observed for binding to recombinant FcepsilonRIalpha. These peptides may prove useful as targets for the identification of antagonists of FcepsilonRIalpha-mediated biological activity. Moreover, our data indicate that FcepsilonRIalpha-mediated activation may involve a novel alpha-chain epitope in an early step of the cell-triggering pathway leading to cellular activation.

The characterization and quantification of antigen-induced Ca2+oscillations in a rat basophilic leukaemia cell line (RBL–2H3)

Using1999 Harcourt Publishers Ltdthe ratiometric Ca2+indicator, indo-1, the antigen-induced increase in intracellular Ca2+concentration ([Ca2+]i) was measured in individual RBL–2H3 cells which had been passively sensitized with monoclonal antibody to the dintrophenyl (DNP) haptenic group. Antigenic stimulation using DNP-human serum albumin conjugate (DNP-HSA) induced concentration-dependent asynchronous Ca2+oscillations, or irregular spikes. To achieve a quantitative comparison of the effects of different concentrations of antigen on changes in [Ca2+]i, the area under the curve (AUC) of Ca2+oscillations in each cell was calculated.The dose–response curve of the calculated AUC is consistent with the bell-shaped dose–response curve for antigen-induced mediator release, depolarization and86Rb+-efflux. Ca2+oscillations induced by antigenic stimulation were abolished by removal of external Ca2+and the subsequent reintroduction of external Ca2+caused their resumption.To investigate the role of Ca2+oscillations in the secretory response, changes in [Ca2+]iinduced by concanavalin A (Con-A), A23187, thapsigargin and NECA were also monitored. Con-A mimicked the response induced by antigen, whilst A23187 and thapsigargin induced a large transient non-oscillatory response. NECA, an adenosine receptor agonist, induced only a small transient rise in [Ca2+]iwithout oscillatory behaviour. Since all these stimuli accept NECA-induced degranulation in these cells, it is suggested that, although Ca2+oscillations are not essential for the initiation of secretion, they probably underlie the in-vivo physiological response of mast cells and basophils to an antigenic challenge. They also seem to enhance the efficacy of the Ca2+signal.

Evidence that IgE Receptor Stimulation Increases Adenosine Release from Rat Basophilic Leukaemia (RBL-2H3) Cells

Adenosine may play a role in asthma by enhancing inflammatory mediator release from lung mast cells. In this study, we investigated whether adenosine is released from cultured rat basophilic leukaemia (RBL-2H3) cells in response to antigen challenge and whether released adenosine enhances mediator release. RBL-2H3 cells closely resemble mucosal mast cells, the most common type of mast cell in lung tissue, and they express adenosine A3receptors (which have been associated with asthma). Measurement of adenosine in RBL-2H3 cell incubation medium was possible if adenosine metabolism was inhibited by EHNA (10 μm; an adenosine deaminase inhibitor) and 5-iodotubericidin (5-IT; 10 μm; an adenosine kinase inhibitor). Basal adenosine concentration increased up to 1.0 μmduring a 90 min incubation; after antigen challenge, adenosine concentration was increased by 0.3–0.4 μmabove basal. Antigen-induced adenosine release ranged from 30–70 nmol/1.25×106cells. Antigen-induced mediator release (β-hexosaminidase and [3H]5-hydroxytryptamine) was increased by APNEA, an adenosine A3receptor agonist (EC50approximately 20 nm) but inhibited by EHNA and 5-IT, despite increased adenosine levels. This inhibition was not blocked by the adenosine A1/A2receptor antagonist DPSPX (5 μm), therefore, it is unlikely to be related to adenosine receptor activation. In conclusion, although our data provide no direct support for a positive feedback effect of adenosine on mast cell mediator release, the observation that IgE receptor stimulation increases adenosine production in cells which express stimulatory A3receptors is consistent with this hypothesis.

The strain difference in the effect of mercuric chloride on antigen-triggered serotonin release from rat mast cells is not mediated via interferon-gamma.

Previous work has shown that in vitro exposure of Brown-Norway (BN) rat peritoneal mast cells to mercuric chloride (HgCl2) causes enhancement of subsequent mediator release induced by cross-linking of surface immunoglobulin E (IgE). This enhancing effect is seen significantly less often with peritoneal cells from Lewis rats. In addition HgCl2 has been shown to suppress interferon (IFN)-gamma production by BN but not Lewis splenocytes. Given that IFN-gamma is known to inhibit mediator release by mast cells, we hypothesized that the strain difference in the effect of HgCl2 on mediator release was mediated via a differential effect on IFN-gamma release from T cells in the mixed peritoneal cell population: IFN-gamma release would be suppressed in the case of the BN rat, releasing the mast cells from inhibition and resulting in the enhancing effect of HgCl2. The aim of the study was to test two predictions of this hypothesis. Exposure of BN rat mast cells to IFN-gamma inhibited subsequent antigen-induced mediator release but did not significantly reduce HgCl2-mediated enhancement of this release. Exposure of Lewis rat mast cells to blocking concentrations of anti-IFN-gamma did not reveal any HgCl2-mediated enhancement of mediator release. These observations provide strong evidence against the hypothesis that the differential effects of HgCl2 on BN and Lewis rat mast cells are mediated via IFN-gamma. In addition the results revealed that BN rat mast cells are significantly more sensitive than Lewis rat mast cells to the inhibitory effects of IFN-gamma on antigen-induced mediator release.

A rationale for the use of nedocromil sodium in the treatment of asthma

A rationale for the use of nedocromil sodium in the treatment of asthma

163 Substance P-induced and antigen-induced release of mediators from guinea pig skin in vitro

163 Substance P-induced and antigen-induced release of mediators from guinea pig skin in vitro

Products of arachidonic acid metabolism and the effects of cyclooxygenase inhibition on ongoing cutaneous allergic reactions in human beings

Background: There have been conflicting reports about the effects of inhibition of arachidonic acid metabolism on early- and late-phase cutaneous reactions. We re-examined this question with a unique nonsteroidal antiinflammatory drug, tenidap sodium. Tenidap sodium has been demonstrated in in vitro studies to inhibit cyclooxygenase, lipoxygenase, and cytokine production (interleukin-1, interleukin-6, tumor necrosis factor-α). Methods: In a double-blind, randomized, crossover study, seven pollen-sensitive subjects ingested tenidap (120 mg, by mouth, daily) and placebo for 9 days with a 3-week washout period between treatments. On the eighth day they underwent allergen skin testing, measurable for up to 12 hours, and on the ninth day they underwent 5-hour skin chamber exposures to allergen and buffer. Chamber fluids were analyzed for cellular content, neutrophil granule protein release, cyclooxygenase and lipoxygenase arachidonic acid metabolites, histamine, and tryptase. Results: Tenidap did significantly inhibit cyclooxygenase metabolites at both antigen and buffer sites but had no effect on histamine, tryptase, lipoxygenase metabolites, or granulocyte infiltration. Neutrophil granule release of lactoferrin was lower at the antigen site during tenidap administration, but there was no reduction of elastase release. Prostaglandin E 2 and leukotriene E 4 increased significantly at antigen sites compared with buffer sites during placebo administration and were the most prominent arachidonic acid metabolites detected. Conclusion: Tenidap, despite inhibiting cyclooxygenase release at antigen sites, had no effect on skin test responses to antigen or on antigen-induced mediator release or granulocyte infiltration. We conclude that cyclooxygenase metabolites are not important in the development of an allergic cutaneous inflammatory response. (J A LLERGY C LIN I MMUNOL 1995;95:742-7.)

Formoterol on airway smooth muscle and human lung mast cells: a comparison with salbutamol and salmeterol

Formoterol, like salbutamol and salmeterol, relaxed isolated preparations of guinea-pig trachea and human bronchus, and inhibited antigen-induced mediator release from human lung fragments in a concentration-related fashion. In each case, these actions were mediated through β 2-adrenoceptors, with formoterol being 50–120-fold more potent than salbutamol, and 2–27-fold more potent than salmeterol. The duration of action of formoterol was longer than that of salbutamol in all preparations, but was markedly shorter than that of salmeterol, whose actions persisted for many hours despite continuous or extensive washing of the tissues. In conscious guinea-pigs, inhaled formoterol, salbutamol and salmeterol all caused dose-related inhibition of histamine-induced bronchoconstriction. Formoterol was again more potent (10–20-fold) than either salbutamol or salmeterol. However, while the actions of a threshold-effective dose of formoterol persisted for less than 3 h, somewhat longer than those of salbutamol (< 1.5 h), an equivalent dose of salmeterol was active for at least 6 h. Therefore, while formoterol is a potent β 2-adrenoceptor agonist in vitro and in vivo, and is consistently longer-acting than salbutamol, its duration of action is markedly shorter than that of salmeterol.

Inhibitory Effect of DS-4574 on Leukotriene- or Antigen-Induced Bronchoconstriction in Guinea Pigs

We studied the leukotriene (LT) antagonistic activity of DS-4574 in vivo and the inhibitory effect of this compound on antigen-induced bronchoconstriction in actively sensitized guinea pigs. Bronchoconstriction induced by LTD4 was inhibited by intravenous and oral treatment with DS-4574 in a dose-dependent manner. Orally administered DS-4574 was also able to inhibit the bronchoconstriction mediated by intravenous administration of LTC4 and E4 and that by endogenous LTs. The inhibitory effect of DS-4574 showed similar potency to those of FPL-55712 and LY171883. In contrast, histamine-, acetylcholine- or 5-hydroxytryptamine-induced bronchoconstriction was not significantly affected by DS-4574. Moreover, DS-4574 given orally or intravenously inhibited antigen-induced bronchoconstriction in actively sensitized guinea pigs and this compound prevented antigen-induced mediator release from actively sensitized guinea-pig lung fragments. The anti-asthmatic effect of this compound appears to be associated with LT antagonism and inhibition of the release of chemical mediators. This study therefore shows DS-4574 to have orally effective LT antagonistic and anti-asthmatic activities. This compound may prove useful in the treatment of bronchial asthma.

Salmeterol: a potent and long‐acting inhibitor of inflammatory mediator release from human lung

1. The effects of salmeterol, a novel long-acting beta 2-adrenoceptor agonist, have been investigated on antigen-induced mediator release from passively sensitized fragments of human lung in vitro. 2. Salmeterol was a potent inhibitor of the release of histamine (-log IC50 = 8.54), leukotriene C4 (LTC4)/LTD4 (-log IC50 = 9.07) and prostaglandin D2 (-log IC50 = 8.81). It was slightly less potent (1-3 fold) than isoprenaline, but significantly more potent (10-35 fold) than salbutamol. 3. Propranolol competitively antagonized the inhibitory effects of salmeterol on histamine release (pA2 = 8.41) and LTC4/LTD4 release, (pA2 = 8.40) indicating an action via beta-adrenoceptors. 4. The inhibitory effects of isoprenaline (20 nM) and salbutamol (200 nM) were removed after washing the lung tissue for 2 h and 4 h respectively. In contrast, the inhibitory effects of salmeterol (40 nM) were much longer-lasting, and were still evident after 20 h. 5. Salmeterol therefore exhibits potent and persistent inhibition of anaphylactic mediator release from human lung. This anti-inflammatory effect may be important for the therapeutic potential of salmeterol in the treatment of bronchial asthma.

Antigen-induced mediator release in primates.

We examined the release of bronchoactive mediators into the airways of allergic primates during the acute response to specific antigen inhalation. Twelve adult male cynomolgus monkeys (Macaca fascicularis) with a naturally occurring respiratory sensitivity to inhaled Ascaris suum extract were anesthetized and intubated for each study. Respiratory system resistance (Rrs) and dynamic lung compliance (CLdyn) were measured before and after antigen inhalation, and the release of mediators into the airways was assessed by bronchoalveolar lavage (BAL). BAL samples were concentrated approximately 5-fold before quantitation of LTC4 and PGD2 by RP-HPLC and radioimmunoassay and histamine by a fluorometric assay. Antigen inhalation resulted in a 40-fold increase in BAL levels of i-LTC4 (1.5 +/- 0.7 to 41.6 +/- 12.7 ng, p less than 0.01), a 10-fold increase in i-PGD2 (2.4 +/- 0.9 to 25.9 +/- 5.5 ng, p less than 0.01), and a 20-fold increase in BAL histamine (1.0 +/- 1.5 to 21.4 +/- 2.3 micrograms, p less than 0.01). Dexamethasone (n = 7) inhibited the antigen-induced increase in BAL i-LTC4 (71 +/- 6%, p less than 0.01) and i-PGD2 (52 +/- 8%, p less than 0.05) while weakly inhibiting histamine release (43 +/- 10%). Indomethacin (n = 7) had a variable effect on i-LTC4 levels (6 +/- 51%), strongly inhibited i-PGD2 (88 +/- 9%, p less than 0.01), and had no effect on histamine release (25 +/- 8%). Pretreatment with iodoxamide tromethamine significantly blocked the release of each mediator, but mepyramine, an H1 antagonist, had no effect on mediator release.(ABSTRACT TRUNCATED AT 250 WORDS)