Germinal center formation and local immunoglobulin E (IgE) production in the lung after an airway antigenic challenge.

Airway inflammation plays a central role in the pathogenesis of asthma. However, the precise contribution of all cell types in the development and maintenance of airway hyperreactivity and histopathology during allergic inflammation remains unclear. After sensitization of mice in the periphery, challenge by multiple intratracheal (i.t.) instillations of ovalbumin (OVA) results in eosinophilia, mononuclear cell infiltration, and airway epithelial changes analogous to that seen in asthma (Blyth, D.I., M.S. Pedrick, T.J. Savage, E.M. Hessel, and D. Fattah. 1996. Am. J. Respir. Cell Mol. Biol. 14:425-438). To investigate further the nature of the cellular infiltrate, lungs from OVA-versus saline-treated mice were processed for histology and immunohistochemistry. One of the most striking features observed was the formation of germinal centers within the parenchyma of the inflamed lungs. In addition, follicular dendritic cells (FDCs) bearing OVA on their plasma membranes appeared and, adjacent to these sites, OVA-specific IgG1-, IgE-, and IgA-producing plasma cells emerged. To confirm that antigen-specific immunoglobulins (Ig) were being produced within the parenchyma, plasma cell number and antibody production were quantitated in vitro after isolation of cells from the lung. These assays confirmed that the isotypes observed in situ were a secreted product. As IgE-dependent mechanisms have been implicated as being central to the pathogenesis of bronchial asthma, airway hyperresponsiveness was evaluated. The mice undergoing lung inflammation were hyperresponsive, while the control group remained at baseline. These data demonstrate that antigen-driven differentiation of B cells via induction of an FDC network and germinal centers occurs in the parenchyma of inflamed lungs. These germinal centers would then provide a local source of IgE-secreting plasma cells that contribute to the release of factors mediating inflammatory processes in the lung.

A transgenic model for listeriosis: role of internalin in crossing the intestinal barrier.

Listeria monocytogenes is responsible for severe food-borne infections, but the mechanisms by which bacteria cross the intestinal barrier are unknown. Listeria monocytogenes expresses a surface protein, internalin, that interacts with a host receptor, E-cadherin, to promote entry into human epithelial cells. Murine E-cadherin, in contrast to guinea pig E-cadherin, does not interact with internalin, excluding the mouse as a model for addressing internalin function in vivo. In guinea pigs and transgenic mice expressing human E-cadherin, internalin was found to mediate invasion of enterocytes and crossing of the intestinal barrier. These results illustrate how relevant animal models for human infections can be generated.

Lipopolysaccharide from Escherichia coli reduces antigen-induced bronchoconstriction in actively sensitized guinea pigs.

Bronchoconstriction (BC) is the main feature of anaphylaxis in the guinea pig. Since LPS induces lung inflammation and antigen-induced BC depends on the endogenous formation of histamine and arachidonate metabolites, we studied whether LPS might modulate antigen-induced BC. Guinea pigs were sensitized subcutaneously with 10 micrograms ovalbumin (OA) on days 0 and 14. LPS (100 micrograms/kg) was injected intravenously on day 21, and daily injections of LPS were continued before the antigenic challenge on day 22, 23, 24, or 25. Intratracheal injection of 100 micrograms OA induced an abrupt and reversible BC. Single or repetitive injections of LPS reduced BC. LPS is likely to reduce the OA-induced BC by affecting the histamine-dependent component of BC, since (a) LPS induced a partial degranulation of lung mast cells; (b) BC is reduced by mepyramine, an histamine receptor antagonist; (c) LPS did not affect BC in mepyramine-treated guinea pigs; (d) LPS reduced histamine release by OA-stimulated guinea pig lungs in vitro. Moreover, the in vitro OA-induced production of arachidonate metabolites was also reduced by LPS. The decreased formation of TXB2 was not only secondary to a reduced release of histamine, since LPS inhibited TXB2 formation in the presence of mepyramine. Finally, the FMLP-induced BC and mediator release were inhibited by LPS, whereas the platelet activating factor-induced pulmonary responses were not. Thus, the protective effect of LPS is not antigen-specific and does not result from a general desensitization. These studies indicate that a single dose of LPS reduces the antigen-induced BC by reducing histamine release from lung mast cells, although a decreased formation of eicosanoids may contribute to the protective effect of LPS.

In vivo neutralization of eosinophil-derived major basic protein inhibits antigen-induced bronchial hyperreactivity in sensitized guinea pigs.

This study examines the effect of purified rabbit antiguinea pig eosinophil-derived major basic protein (MBP) Ig on antigen-induced bronchial hyperreactivity to inhaled acetylcholine in aerosol-sensitized guinea pigs. Ovalbumin inhalation by sensitized guinea pigs induced a rise in the numbers of eosinophils and in the levels of MBP in the bronchoalveolar lavage fluid, which peaked at 24 h and resolved at 72 h. Antigen-challenged animals exhibited bronchial hyperreactivity to inhale acetylcholine at 72 h, but not at 6 or 24 h. The intranasal administration of 200 microliter of purified rabbit anti-guinea pig MBP Ig, at 2.5 mg/ml, but not of the control preimmune rabbit Ig, 1 h before and 5 h after ovalbumin inhalation suppressed bronchial hyperreactivity to acetylcholine at 72 h without affecting the number of eosinophils accumulating in the bronchoalveolar lavage fluid. These findings indicate that antigen challenge in sensitized guinea pigs is followed by early eosinophil infiltration and activation within the airways and by late bronchial hyperreactivity. Neutralization of endogenously secreted MBP by a specific antiserum prevented antigen-induced bronchial hyperreactivity, suggesting that eosinophil degranulation plays an important role in the alterations of bronchopulmonary function in the guinea pig.

Structure-activity relationship in PAF-acether. 2. rac-1-O-Octadecyl-2-O-acetyl-3-O-[gamma-(dimethylamino)propyl]glycerol .

Two products without phosphoryl groups, 1-O-octadecyl-2-O-acetyl-3-O-[gamma-(dimethylamino)propyl]glycerol and its quaternary salt, were synthesized from 1-O-octadecyl-2-O-benzylglycerol. In comparison with PAF-acether, they lost aggregating and bronchoconstrictive activities and did not show any antagonistic effects.

Role of platelets in aspirin-sensitive bronchoconstriction in the guinea-pig; interactions with salicylic acid.

1 The bronchoconstriction caused in the guinea-pig by arachidonic acid (AA), bradykinin, adenosine diphosphate (ADP) and adenosine triphosphate (ATP) was correlated with effects on platelets. ATP and ADP produced a brief thrombocytopenia and AA a more prolonged one. Bradykinin had no effect on platelets.2 Aspirin inhibited bronchoconstriction and thrombocytopenia produced by AA and part of the bronchoconstriction produced by ATP, but had no effect against ADP. Thrombocytopenia produced by ADP and ATP was not affected by aspirin or indomethacin.3 Platelet depletion by antiserum prevented bronchoconstriction in response to ADP and to ATP, but not in response to bradykinin or to AA, showing that platelets are not involved in aspirin-sensitive bronchoconstriction. Infusions of ADP reduced bronchoconstriction and thrombocytopenia in response to ADP itself and to ATP, but not to AA. Bronchoconstriction by ADP or ATP involves an action on platelets. Only that due to ATP is partially dependent on the activity of prostaglandin synthetase.4 ATP induced aggregation in vitro in guinea-pig platelet-rich plasma (PRP). Rabbit PRP responded only when ATP was first incubated with guinea-pig plasma. The aggregating compound formed was probably ADP, since it was destroyed by apyrase. Its formation was not inhibited by aspirin or indomethacin, indicating that aspirin inhibits ATP-induced bronchoconstriction by a different mechanism.5 The aggregating effect of ATP on guinea-pig platelets was inhibited by concentrations of apyrase that block ADP-induced aggregation, and potentiated by lower concentrations of apyrase.6 Adenosine 5'-tetraphosphate did not aggregate platelets in vivo or in vitro. In vitro aggregation occurred when apyrase was added, suggesting transformation into ADP. Adenosine 5'-tetraphosphate and apyrase inhibited aggregation due to ADP, but failed to affect that due to AA. This suggests that aggregation involving products of prostaglandin synthesis does not require ADP.7 Salicylic acid did not interfere with bronchoconstriction or aggregation due to AA, but prevented inhibition by aspirin when the weight ratio, salicylic acid:aspirin was 4:1. Salicyclic acid may be useful in studies of potential inhibitors of thromboxane A2 synthesis and of thromboxane A2-dependent processes in vivo and in vitro.

Blockade by methylation inhibitors of the anaphylactic response of guinea-pig lung strips.

1. The combination of two methylation inhibitors 3-deazaadenosine (10(-4) or 4 x 10(-4) M) plus L-homocysteine (2 x 10(-4) M) caused a time-dependent inhibition of antigen-induced contraction, formation of thromboxane B2 (TxB2) and release of histamine from lung parenchyma strips taken from guinea-pigs actively sensitized with ovalbumin (OA). 2. The methylation inhibitors also prevented the lung strip contractions induced by the mediators platelet-activating factor (Paf-acether, 10(-6) M), leukotriene D4 (LTD4, 10(-8) and 3 x 10(-8) M), and in part to arachidonic acid (10(-6) and 10(-5) M), under conditions where the contractions to histamine (10(-6)-10(-4) M) were virtually unaffected. 3. TxB2 formation induced by these mediators or by OA was more affected by the methylation inhibitors than the lung strip contractions, indicating that prostaglandin formation is more sensitive to these inhibitors than the myotropic activity. In contrast, the suppressive effect of the methylation inhibitors on histamine secretion by parenchyma lung strips induced by OA followed the inhibition of the contraction. 4. These results show that inhibitors of methyltransferases interfere with the myotropic responses and with the release of mediators by actively sensitized guinea-pig lung strips stimulated with antigen, and suggest a major role for a methylation process in mediating the contraction of and mediator release by the lung parenchyma.

The effect of cetirizine on antigen-dependent leucopenia in the guinea-pig.

1. Intravenous administration of ovalbumin (1 mg kg-1) to guinea-pigs that had previously been injected with 3.5 x 10(9) platelets from actively sensitized animals induced an approximately 40% decrease in the number of circulating leucocytes 30-60 min later, whereas the number of platelets was not affected. 2. In contrast, there was no change in the leucocyte number following antigen challenge of guinea-pigs that had received platelets from non-immunised animals. 3. This platelet-dependent leucopenia was inhibited by prior treatment of the recipient animal with cetirizine (10-30 mg kg-1, i.v.). Terfenadine (50 mg kg-1, p.o.) and mepyramine (2 mg kg-1, i.v.) were completely inactive in this respect. All doses of anti-histamines were used at concentrations which completely inhibited the bronchoconstriction to an i.v. injection of 5 micrograms kg-1 of histamine. 4. The site of action of cetirizine is most likely to be the platelet as leucopenia induced by the neutrophil agonists leukotriene B4 (LTB4) (30 ng kg-1) and platelet activating factor (PAF) (30 ng kg-1) were not modified by cetirizine treatment. 5. In these experiments, we failed to support a role for lipoxygenase products as mediators of the platelet-dependent leucopenia, as the selective lipoxygenase inhibitor BWA4C (50 mg kg-1, p.o.) was ineffective. 6. Our present results confirm and extend previous data demonstrating that antigen stimulated platelets can induce leucopenia in non-immunised animals and this can be inhibited by the anti-allergic agent, cetirizine, by an action which is probably unrelated to its anti-histamine properties. The precise nature of the platelet derived factor(s) and their target of action remains to be determined.

Histamine and leukotriene-independent guinea-pig anaphylactic shock unaccounted for by Paf-acether.

Ovalbumin induced dose-dependent contractions of lung parenchyma strips from sensitized guinea-pigs, whereas Paf-acether (1-alkyl-2-acetyl-sn-glycero-3-phosphoryl choline), a potential mediator of immediate hypersensitivity, induced a single contraction, followed by specific desensitization to a second exposure. Lung strips desensitized to Paf-acether contracted to ovalbumin as did non-desensitized controls, even in the presence of inhibitors of other mediators of anaphylaxis. Contractions by Paf-acether and by ovalbumin were reduced by nordihydroguaiaretic acid (NDGA) and by the phospholipase A2 inhibitor p-bromophenacyl bromide (0.1-0.3 mM). Three other anti-lipoxygenase agents (diethylcarbamazine, 5 mM; eicosatetraynoic acid and BW755c, 0.1 mM), reduced the contractions by ovalbumin but also those due to acetylcholine, indicating non-specific effects. Neither the anti-allergic compound sodium cromoglycate (3 mM) nor the anti-leukotriene agent, FPL 55712 (0.01 mM), inhibited the contractions by ovalbumin or by Paf-acether. A sensitized strip stimulated with ovalbumin released substances which contracted a non-sensitized strip mounted in the same organ bath. The contractions of the non-sensitized strip were abolished by FPL 55712 (0.01 mM), by NDGA and BW755c, (0.1 mM), whereas those of the sensitized one were unaffected. Leukotrienes are formed by the lung strips during shock but alone, they do not explain the contractile activity. The intravenous administration of ovalbumin (1 mg kg-1) led to bronchoconstriction and thrombocytopenia, which were not modified by the anti-leukotriene substance FPL 55712 nor by aspirin. Bronchoconstriction was suppressed if FPL 55712 was used in combination with aspirin (20 mg kg-1), mepyramine and methysergide (200 micrograms kg-1 of either). Pretreatment of the guinea-pigs with propranolol reduced this inhibition to approximately 60%. In no instance was thrombocytopenia prevented. In vitro contractions of the actively sensitized lung strip are not fully accounted for by histamine, FPL 55712-inhibitable leukotrienes or Paf-acether, whereas in systemic anaphylaxis histamine and leukotrienes (inhibited respectively by mepyramine and by FPL 55712) have a significant role.

Limited interference of specific Paf antagonists with hyper-responsiveness to Paf itself of lungs from actively sensitized guinea-pigs.

1. The interference of various platelet-activating factor (Paf) antagonists with Paf- and antigen-induced effects in isolated lungs from actively sensitized guinea-pigs was investigated. 2. WEB 2086 and BN 52021, two antagonists structurally unrelated to Paf, at concentrations 10-100 fold above those exerting a potent and selective inhibition of the effects of Paf in lungs from non-immunized guinea-pigs, failed to inhibit bronchoconstriction and mediator release evoked by the phospholipid when administered into lungs from actively sensitized animals. 3. In contrast to WEB 2086 and BN 52021, antagonists such as Ro 19-3704 and Ro 19-1400, structurally related to the Paf molecule, at concentrations which abrogate the effects of Paf in lungs from non-immunized guinea-pigs, inhibited bronchoconstriction and release of histamine and leukotriene-like material evoked by the intra-arterial administration of Paf into lungs from actively sensitized animals. 4. Ro 19-3704 and Ro 19-1400 also inhibited markedly the release of leukotriene C4 (LTC4)-like material and, to a smaller extent, the histamine secretion induced by 10 micrograms arachidonic acid. 5. CV 6209, another Paf antagonist structurally related to the phospholipid, failed to antagonize its bronchopulmonary and secretory effects in sensitized lungs. 6. All the antagonists used, irrespective of their ability to interfere or not with bronchoconstriction and mediator release triggered by Paf, suppressed oedema formation as measured by the increase in lung wet weight induced by either Paf or ovalbumin. 7. Our results indicate that: (i) the increase in vascular permeability and the subsequent oedema formation on one hand and the bronchopulmonary effects of Paf on the other hand are mediated by different mechanisms; and (ii) active sensitization provokes a marked modification of the lung reactivity to Paf.

The platelet-independent release of thromboxane A2 by Paf-acether from guinea-pig lungs involves mechanisms distinct from those for leukotriene.

Intra-arterial injections of platelet-activating factor (Paf-acether, 10-300 ng) to the perfused guinea-pig lung induced a dose-related bronchoconstriction, followed by contraction of the rat aorta superfused with the lung effluent, indicating the release of thromboxane A2 (TXA2) activity. These effects were matched with injections of bradykinin (Bk) at 100-1000 ng, leukotriene C4(LTC4) at 10-300 ng or arachidonic acid (AA) at 30-300 micrograms. Repeated doses of Paf-acether led to a specific desensitization of the release of TXA2, under conditions where Bk, LTC4 and arachidonic acid retained their ability to release TXA2. Bronchoconstriction and the release of TXA2 induced by Paf-acether were suppressed when the lungs were perfused with acetylsalicylic acid, but not with salicylic acid. The phospholipase A2 inhibitor, p-bromophenacyl bromide suppressed the release of TXA2 by Bk, but did not interfere with its formation from AA, nor with its release with Paf-acether and LTC4. The lipoxygenase inhibitor, nordihydroguaiaretic acid, inhibited to a similar extent the release of TXA2 by Bk, LTC4 and Paf-acether but also reduced directly the formation of TXA2 from arachidonic acid, invalidating its use as a specific antilipoxygenase agent. The leukotriene C4/D4 antagonist, FPL 55712, suppressed the TXA2 releasing effects of LTC4, and was completely inactive against Paf-acether, Bk or arachidonic acid. The aerosol of Paf-acether was tested in the anaesthetized guinea-pig and resulted in bronchoconstriction, unaccompanied by thrombocytopenia. Unlike bronchoconstriction induced by intravenous Paf-acether, which is refractory to cyclo-oxygenase inhibitors, the effects of the aerosol were suppressed by aspirin. Platelet depletion, which blocks the intravenous effects of Paf-acether, failed to interfere with those of the aerosol. Paf-acether induced a marked contraction of the superfused guinea-pig isolated parenchyma lung strip, which was followed by total and irreversible desensitization to itself. The contractile effect was not inhibited by aspirin or indomethacin, atropine, mepyramine, methysergide, phenoxybenzamine or propranolol, indicating that cyclo-oxygenase products, cholinergic stimuli, histamine, 5-hydroxytryptamine and catecholamine mechanisms are not involved. Our results indicate that Paf-acether interacts with pulmonary sites distinct from those for Bk, LTC4 or AA, since no cross-desensitization between Paf-acether and the other agonists was noted, p-bromophenacyl bromide inhibited Bk only and FPL 55712 inhibited only LTC4. The phospholipase A2 involved with the release of the arachidonate needed for the formation of TXA2 by Paf-acether or LTC4-stimulated lungs may differ from the enzyme accounting for its formation by Bk. The cellular sites with which Paf-acether interacts may also be distinct and less readily accessible to p-bromophenacyl bromide.

The booster injection of antigen during active sensitization of guinea-pig modifies the anti-anaphylactic activity of the PAF antagonist WEB 2086.

1. Serum IgG levels of sensitized guinea-pigs bled at various times after the booster injection were evaluated and its capacity to sensitize passively lung strips from normal guinea-pigs assessed. Following the booster injection, both serum IgG and the ability to sensitize passively lung strips increased during the first week and decreased slowly thereafter. 2. The PAF antagonist WEB 2086 (3 mg kg-1, i.v.) blocked the anaphylactic bronchoconstriction induced by intravenous administration of ovalbumin (1 mg kg-1) when guinea-pigs were challenged 2 and 4 days after the booster injection, but became ineffective when tested in guinea-pigs challenged 7, 28 and 56 days after the booster injection. 3. The ability of WEB 2086 to reduce anaphylactic bronchoconstriction of guinea-pigs challenged 2 and 4 days after the booster injection was unrelated to either the selective involvement of one type of immunoglobulin, low IgG titres in sera or a reduced sensitizing capacity. 4. The booster injection, which accounts for the loss of efficacy of WEB 2086 from the fourth day thereafter, probably operates as a PAF-independent inflammatory challenge. 5. The protocol for immunisation and the day of experiment after the booster injection determines the sensitivity of the anaphylactic bronchoconstriction to inhibition of PAF antagonists.

Guinea-pig treatment with pertussis toxin suppresses macrophage-dependent bronchoconstriction by fMLP and fails to inhibit the effects of PAF.

1. Bronchoconstriction and thromboxane B2 (TxB2) release following the intra-tracheal administration of the secretagogue N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) to lungs from pertussis toxin-treated guinea-pigs in vivo and in vitro were inhibited as compared to saline-treated animals, under conditions where the responses to PAF were modified less effectively. 2. The cell target accounting for bronchoconstriction by fMLP and for inhibition by pertussis toxin is located in the airways and is probably the alveolar macrophage. Indeed (a) fMLP-induced superoxide anions and TxB2 formation by alveolar macrophages were inhibited by pertussis toxin given in vivo; (b) Gi proteins of membranes from alveolar macrophages were ADP-ribosylated in vivo by pertussis toxin and (c) bronchoconstriction and TxB2 release in response to the intra-tracheal administration of fMLP to lungs from pertussis toxin-treated animals were restored when alveolar macrophages from control guinea-pigs were transferred into the airways of pertussis toxin-treated animals before lung isolation. 3. Pertussis toxin administered to guinea-pigs in vivo, reduced the subsequent TxB2 formation and superoxide anion release by alveolar macrophages stimulated with PAF, but failed to inhibit PAF-induced bronchoconstriction. 4. Formation of TxB2 by alveolar macrophages following the intra-tracheal administration of fMLP accounts for bronchoconstriction and requires pertussis toxin-sensitive Gi proteins. PAF operates via a different mechanism, which is independent of Gi-like protein and involves mediators other than TxB2 and superoxide anions.

Interference of BN 52021, an antagonist of PAF, with different forms of active anaphylaxis in the guinea-pig: importance of the booster injection.

1. BN 52021, an antagonist of platelet activating factor (PAF), was inactive against bronchoconstriction in guinea-pigs sensitized with low amounts of ovalbumin (OA) injected twice, at a 14 day interval and challenged i.v. 7 days later. 2. Serum IgG titers increased for 7 weeks after the booster injection at day 14 and returned to low levels at day 96. 3. Administered by the intratracheal (i.t.) route at 1 mg, BN 52021 failed to inhibit bronchoconstriction induced by the i.t. administration of OA to guinea-pigs tested 7, 28, 56 and 84 days after the booster injection, even when the titers of circulating IgG had declined with time. BN 52021 was also inactive against bronchoconstriction in guinea-pigs boosted at day 98 and tested 7 days later and against contractions and thromboxane (Tx) B2 and histamine release induced by OA-challenged parenchymal lung strips from the boosted guinea-pigs. 4. Sensitized unboosted guinea-pigs displayed reduced IgG serum titers. Used 21 or 70 days after the sensitizing injection, they did develop bronchoconstriction upon the i.t. instillation of OA, which was blocked by BN 52021. The latter also inhibited OA-induced contractions of lung parenchymal strips from these unboosted guinea-pigs. 5. When boosted and non-boosted guinea-pigs received OA i.t. and bronchoalveolar lavage fluid was collected 10 min later, the number of eosinophils increased markedly in boosted, but not in non-boosted guinea-pigs. 6. The booster injection of antigen thus modifies the response of the lung and PAF appears to be relevant for antigen-induced bronchoconstriction in unboosted animals, but loses its major role following the booster injection.

Effect of human recombinant interleukin-5 on in vitro responsiveness to PAF of lung from actively sensitized guinea-pigs.

1. The intra-tracheal (i.t.) administration of human recombinant interleukin-5 (rhIL-5; 100 or 300 ng) to isolated perfused lungs from guinea-pigs actively sensitized to ovalbumin induced an increased bronchoconstriction and release of thromboxane A2 (TXA2) and histamine into the lung effluent following the subsequent (10 min) intra-arterial injection of platelet-activating factor (PAF). Lung responses to 5-hydroxytryptamine were unaffected by rhIL-5. 2. Hyperresponsiveness to PAF was observed when the lungs were obtained from guinea-pigs used 2 or 7 days after a booster injection of the antigen and, to a lower extent, when they were from animals sensitized by a single antigen administration. By contrast, rhIL-5 did not modify the responses to PAF of lungs from passively sensitized or from adjuvant-treated guinea-pigs, suggesting that immunological stimulation is required to allow the expression of synergism between rhIL-5 and PAF. 3. Guinea-pigs killed 2 and 7 days after the booster injection of the antigen exhibited a marked increase in the number of eosinophils in the bronchoalveolar lavage fluid (BAL), as compared to non-sensitized animals. 4. Our results demonstrate that rhIL-5 and PAF act synergistically to induce enhanced bronchoconstriction and mediator release exclusively when lungs are obtained from guinea-pigs sensitized once to ovalbumin and then boosted. Since recruitment of eosinophils into the airways and the development of hyperresponsiveness to PAF are concomitant, it is suggested that eosinophils are the target cells for interaction between rhIL-5 and PAF.

Anaphylactic bronchoconstriction in BP2 mice: interactions between serotonin and acetylcholine.

1. Immunized BP2 mice developed an acute bronchoconstriction in vivo and airway muscle contraction in vitro in response to ovalbumin (OA) and these contractions were dose dependent. 2. Methysergide or atropine inhibited OA-induced bronchoconstriction in vivo and airway muscle contraction in vitro. 3. Neostigmine potentiated the OA-induced bronchoconstriction in vivo and airway muscle contraction in vitro of BP2 mice. This potentiation was markedly reduced by the administration of methysergide or atropine and when the two antagonists were administered together, the responses were completely inhibited. 4. Neostigmine also potentiated the serotonin (5-HT)- and acetylcholine (ACh)-induced bronchoconstriction and this potentiation was significantly reversed by atropine. 5. These results indicate that OA provokes a bronchoconstriction in immunized BP2 mice by stimulating the release of 5-HT, which in turn acts via the cholinergic mediator, ACh.

Inhibition by the immunosuppressive agent FK-506 of antigen-induced airways eosinophilia and bronchial hyperreactivity in mice.

1. The effect of the immunosuppressive agent, FK-506, an allergen-induced airways eosinophilia and bronchial hyperreactivity (BHR) in hyper IgE mice (BP2 selection) was investigated. 2. Administration of FK-506 at 2 mg kg-1 s.c., 1 h before and 5 h after the first four ovalbumin challenges, reduced the recruitment of eosinophils into the bronchoalveolar lavage fluid (BALF) from 1.36 +/- 0.22 x 10(5) to 0.53 +/- 0.24 x 10(5) cells ml-1 (n = 5-6, P < 0.05; 60% inhibition), inhibited by 80% BHR in response to i.v. 5-HT and practically suppressed BHR in response to inhaled methacholine. 3. The antigen-induced interleukin (IL)-5 formation in the BALF and serum was inhibited by FK-506 by 75% in both instances. 4. FK-506 failed to modify the bronchoconstriction in BP2 mice, suggesting that different mechanisms are involved in acute bronchoconstriction and BHR. 5. The increased number of CD4+, CD8+, CD3+ T lymphocytes in the BALF to antigen-challenged mice was unaffected by FK-506. 6. These findings indicate that antigen-induced in vivo IL-5 release and eosinophil, but not T-cell, infiltration into the bronchial lumen of sensitized BP2 mice are targets for the anti-allergic activities of FK-506.

Effect of cyclo-oxygenase inhibitors and modulators of cyclic AMP formation on lipopolysaccharide-induced neutrophil infiltration in mouse lung.

1. The adult respiratory distress syndrome (ARDS) is an acute lung inflammation developed after direct or indirect contact with pathogenic agents. In the present study, a mouse model was developed to mimic this condition using aerosolized bacterial lipopolysaccharide (LPS) and to investigate the mechanisms involved in the lung inflammatory response. 2. Inhalation of LPS led to a time and dose-dependent increase in tumour necrosis factor-alpha (TNF-alpha) production and neutrophil recruitment into the bronchoalveolar lavage fluid (BALF) of Balb/c mice. Under the same conditions, neutrophil infiltration was also found in the BALF of the LPS-sensitive mouse strain C3H/HeN, but was absent in the LPS-resistant strain C3H/HeJ. Intranasal administration of murine recombinant TNF-alpha also triggered neutrophil recruitment. 3. One hour after inhalation of LPS, half of the maximal level of TNF-alpha was measured in the BALF, but only a few neutrophils were detected at this time. The peak TNF-alpha concentration was reached at 3 h, when the neutrophil amount started to increase. At 24 h, maximal neutrophil number was found in the BALF and TNF-alpha was no longer present. 4. Pretreatment of mice under different experimental conditions demonstrated that: (a) cycloheximide almost completely blocks both neutrophil recruitment and TNF-alpha production; (b) anti TNF-alpha antibodies block neutrophil recruitment; (c) indomethacin or aspirin enhance by two fold neutrophil recruitment; (d) indomethacin significantly increases TNF-alpha production 1 h after inhalation of LPS; (e) dibutyryl cyclic AMP and prostaglandin E2 (PGE2) block both neutrophil recruitment and TNF-alpha production. 5. It is concluded that aerosolized LPS in mice triggers an acute lung inflammation which can be used as a potential model of inhalational ARDS and that, strategies leading to the elevation of cyclic AMP levels in vivo can be effective in modulating LPS-induced TNF-alpha synthesis and neutrophil recruitment.