ovalbumin and verlukast

The multidrug resistance protein 4 (Mrp4) is an ATP-binding cassette transporter that is capable of exporting the second messenger cAMP from cells, a process that might regulate cAMP-mediated anti-inflammatory processes. However, using LPS- or cigarette smoke (CS)-inflammation models, we found that neutrophil numbers in the bronchoalveolar lavage fluid (BALF) were similar in Mrp4(-/-) and Mrp4(+/+) mice treated with LPS or CS. Similarly, neutrophil numbers were not reduced in the BALF of LPS-challenged wt mice after treatment with 10 or 30 mg/kg of the Mrp1/4 inhibitor MK571. The absence of Mrp4 also had no impact on the influx of eosinophils or IL-4 and IL-5 levels in the BALF after OVA airway challenge in mice sensitized with OVA/alum. LPS-induced cytokine release in whole blood ex vivo was also not affected by the absence of Mrp4. These data clearly suggest that Mrp4 deficiency alone is not sufficient to reduce inflammatory processes in vivo. We hypothesized that in combination with PDE4 inhibitors, used at suboptimal concentrations, the anti-inflammatory effect would be more pronounced. However, LPS-induced neutrophil recruitment into the lung was no different between Mrp4(-/-) and Mrp4(+/+) mice treated with 3 mg/kg Roflumilast. Finally, the single and combined administration of 10 and 30 mg/kg MK571 and the specific breast cancer resistance protein (BCRP) inhibitor KO143 showed no reduction of LPS-induced TNFα release into the BALF compared to vehicle treated control animals. Similarly, LPS-induced TNFα release in murine whole blood of Mrp4(+/+) or Mrp4(-/-) mice was not reduced by KO143 (1, 10 µM). Thus, BCRP seems not to be able to compensate for the absence or inhibition of Mrp4 in the used models. Taken together, our data suggest that Mrp4 is not essential for the recruitment of neutrophils into the lung after LPS or CS exposure or of eosinophils after allergen exposure.

Topics: Adenosine; Allergens; Animals; Asthma; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Bronchoalveolar Lavage Fluid; Cyclic AMP; Cytokines; Diketopiperazines; Eosinophils; Heterocyclic Compounds, 4 or More Rings; Lipopolysaccharides; Lung; Mice; Multidrug Resistance-Associated Proteins; Neutrophils; Ovalbumin; Phosphodiesterase 4 Inhibitors; Propionates; Pulmonary Disease, Chronic Obstructive; Quinolines; Rolipram; Smoking; Th2 Cells; Time Factors

Cysteinyl leukotrienes are potent inflammatory molecules playing a major role in asthma. The involvement of these mediators in hypersensitivity in mice is not well known. This study aimed at elucidating their implication by using MK-571, a cysLT(1)receptor antagonist. Mice were sensitized with a suspension of ovalbumin (8 microg) adsorbed to alum (2 mg) and were challenged with an aerosolized ovalbumin solution (0.5%). Inflammatory cell infiltration in the bronchoalveolar lavage (mostly eosinophils) following antigen challenge was inhibited by dexamethasone (0.1, 1 and 5 mg kg(-1)s.c.) and MK-571 (1, 10, 100 mg kg(-1)i.v.) in a dose-dependent manner. Maximal inhibition was 95% with 5 mg kg(-1)dexamethasone and 90% with 100 mg kg(-1)MK-571. When injected together they showed an additive inhibitory effect on eosinophil infiltration. Bronchial hyperreactivity, measured by the increased pulmonary insufflation pressure to carbachol injections, was also inhibited dose-dependently by MK-571. The EC(50)values for carbachol were of 22.39+/-1.12 microg kg(-1)in sensitized and challenged animals that did not receive MK-571 and increased to 43.65+/-1.10, 50.12+/-1.15 and 83.18+/-1.16 microg kg(-1)in animals treated with 1, 10 and 100 mg kg(-1)MK-571 respectively. Lung microvascular leakage (as measured by Evans blue extravasation) induced by antigen bronchoprovocation was reduced by 22% after treatment with 10 mg kg(-1)MK-571. All these inhibitory effects of MK-571 suggest a role for leukotriene D(4)in this animal model of allergic asthma.

Topics: Animals; Asthma; Bronchial Hyperreactivity; Bronchial Provocation Tests; Bronchoalveolar Lavage Fluid; Capillary Permeability; Dexamethasone; Disease Models, Animal; Drug Therapy, Combination; Leukotriene Antagonists; Leukotriene D4; Male; Membrane Proteins; Mice; Mice, Inbred BALB C; Ovalbumin; Propionates; Pulmonary Eosinophilia; Quinolines; Receptors, Leukotriene

We have recently demonstrated that tissue resistance increases during the early response (ER) to antigen challenge in sensitized Brown-Norway rats. The purpose of the present study was to investigate the in vitro airway and tissue responses to antigen and the involvement of the potential mediators serotonin (5-HT) and leukotriene D4 (LTD4). We sensitized Brown-Norway rats with ovalbumin (OA) and subsequently challenged bronchial rings and subpleural parenchymal strips with OA in the organ bath. In selected experiments tissues were incubated with methysergide (a 5-HT receptor antagonist), ketanserin (a 5-HT2 receptor antagonist), MK-571 (a LTD4 receptor antagonist), or MK-886 (5-lipoxygenase inhibitor) prior to challenge. Both bronchial rings and parenchymal strips constricted in response to OA. Methysergide and ketanserin completely inhibited OA-induced constriction of bronchial rings. The effect of MK-571 was not significant, whereas MK-886 partially blocked OA-induced bronchial constriction, suggesting a potential role for LTC4 in antigen-induced airway constriction. In parenchymal strips, methysergide, ketanserin, MK-571, and MK-886 all partially inhibited the OA response, whereas the combinations of methysergide and MK-571 or ketanserin and MK-886 completely ablated the response. These data suggest that both bronchial rings and parenchymal strips constrict after OA challenge but that the relative contributions of 5-HT and LTD4 to the allergic response in central airways and parenchymal tissues differ.

Topics: Airway Resistance; Animals; Bronchi; Bronchial Provocation Tests; Bronchoconstriction; Indoles; Ketanserin; Leukotriene D4; Lipoxygenase Inhibitors; Lung; Methysergide; Ovalbumin; Propionates; Quinolines; Rats; Rats, Inbred BN; Receptors, Immunologic; Respiratory Hypersensitivity; Serotonin

The ovalbumin (OA)-sensitized Brown Norway rat (BN) demonstrates early-response (ER) and late-response (LR) allergic bronchoconstriction. To determine whether these responses could be replicated in vitro, we studied lung explants from 8-wk-old male BN rats (wt: 239 +/- 28 g), of which 19 were sensitized to OA (test) and 16 served as controls. Two weeks after sensitization, the animals' lungs were removed, filled with a 1% (wt/vol) agarose-containing solution at 37 degrees C, and cooled to 4 degrees C. Transverse slices (0.5 to 1.0 mm thick) were cut and cultured overnight. Airways were visualized with an inverted microscope and baseline images were obtained with a video camera. To study the ER, 40 airways from 15 test rats and 29 airways from 10 control rats were challenged with 2 micrograms OA and imaged each minute for 10 min. To study the LR, 40 airways from 12 test rats and 44 airways from 12 control rats were challenged with 2 micrograms OA and imaged each hour for 8 h. The maximal response (MR) for each airway was defined as the percent of airway closure. The ER and LR were both defined as an MR > or = mean + 2 SD of the controls. An ER occurred in 38 of 40 test and 2 of 29 control airways (mean MR: 42 +/- 24% versus 4 +/- 3%, p < 0.001), and was completely blocked by methysergide pretreatment in 13 airways.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Airway Resistance; Animals; Asthma; Bronchial Provocation Tests; Bronchodilator Agents; Constriction, Pathologic; Disease Models, Animal; Drug Hypersensitivity; Hypersensitivity, Delayed; Hypersensitivity, Immediate; Immunoglobulin E; In Vitro Techniques; Leukotriene D4; Male; Methysergide; Ovalbumin; Premedication; Propionates; Quinolines; Rats; Serotonin; Time Factors

The purpose of the study was to investigate whether allergen-induced hyperresponsiveness to methacholine and an increase in airway smooth muscle (ASM) in Brown Norway (BN) rats could be mediated by LTD4, an important mediator of allergic airway responses. Male BN rats, 8 to 12 wk of age, were sensitized with ovalbumin (OA). Rats were exposed 2 wk later to aerosols of saline (n = 6), OA (n = 8), or OA after pretreatment with the LTD4 antagonist MK-571 (2 mg/kg intraperitoneally, n = 9), on six occasions at 5-day intervals. Airway responsiveness to methacholine (the concentration required to double pulmonary resistance, EC200 RL) was measured immediately before the first aerosol exposure and 2 days after the last exposure. ASM was quantitated by morphometry, and areas were standardized for size using the epithelial basement membrane length (BM). Following OA challenges EC200 RL decreased from 6.5 to 3.1 mg/ml (p < 0.05) but did not change significantly after saline or OA exposures in MK-571-pretreated animals. ASM/BM2 in the large airways was significantly greater, 3.41 +/- 0.19 x 10(-3), after OA compared with 2.35 +/- 0.22 x 10(-3) for saline exposures (p < 0.01). The ASM/BM2 after OA exposures but with MK-571 pretreatment (2.75 +/- 0.25 x 10(-3)) was intermediate in value. The results indicate that both the increase in airway responsiveness and the increase in ASM following repeated antigen exposures appear to be mediated predominantly by LTD4.

Topics: Airway Resistance; Animals; Basement Membrane; Bronchial Hyperreactivity; Bronchial Provocation Tests; Bronchodilator Agents; Immunization; Lung; Male; Methacholine Chloride; Muscle, Smooth; Ovalbumin; Pressure; Propionates; Pulmonary Ventilation; Quinolines; Rats; Receptors, Immunologic; Respiratory Muscles; SRS-A; Time Factors

Male Dunkin Hartley guinea pigs, actively sensitized to ovalbumin (OA) and pretreated with mepyramine (1 mg/kg i.p.), were challenged with OA as an aerosol. Histological analysis of the lung for eosinophils (EOs) showed significant accumulations in the submucosa of the airways (8 h: 1.477.3 +/- 43 EOs/mm2 airway, n = 3, p less than 0.01), after antigen challenge compared to saline control (478.6 +/- 104 EOs/mm2 airway, n = 4). Pretreatment with both mepyramine and cimetidine (10 mg/kg i.p.) did not affect this response. Aerosolization of OA to unsensitized guinea pigs resulted in no significant accumulation of EOs (360.2 +/- 49 EOs/mm2 airway, n = 4) when compared with the saline-challenged sensitized control group. Pretreatment with the leukotriene (LT)D4 receptor antagonist MK-571 (1 mg/kg p.o.) significantly inhibited the OA-induced EO migration (95 +/- 5% inhibition, n = 5, p less than 0.01) compared to vehicle in the presence of mepyramine and cimetidine, while indomethacin (3 mg/kg p.o., n = 4) had no effect. Aerosolization of synthetic LTC4 (0.3-30 micrograms/ml) resulted in a dose-dependent migration of EOs into the submucosal layer over 8 h, reaching significance at the 10-micrograms/ml dose, with comparable results obtained with LTD4. Pretreatment of animals with MK-571 (1 mg/kg p.o.) before LTC4 and LTD4 aerosol results in inhibition of the response in both cases, suggesting that this effect may be mediated through the LTD4 receptor. We conclude that peptide LTs produce eosinophilia in the airways of the guinea pig.

Topics: Animals; Bronchial Hyperreactivity; Cell Movement; Cimetidine; Eosinophils; Guinea Pigs; Indomethacin; Leukotrienes; Male; Ovalbumin; Propionates; Pyrilamine; Quinolines; Receptors, Immunologic; Receptors, Leukotriene

Chemotaxis of guinea-pig eosinophils in vivo has been studied in the guinea-pig conjunctiva. Guinea-pig eosinophils were labelled with 111In oxine and injected i.v. into recipient animals. Circulating radioactivity remained constant over a period of 4 h but dropped transiently in response to a bolus injection (1 nmol) of platelet activating factor (PAF), suggesting that the 111In-labelled eosinophils had retained their responsiveness to PAF. Leukotriene D4 (LTD4, 1 nmol/eye) and PAF (10 nmol/eye), but not histamine (5 nmol/eye), induced a significant 2.5-fold increase in conjunctival radioactivity, a measure of eosinophil chemotaxis in vivo, after 17 h. Antigen challenge (250 micrograms/eye) in ovalbumin-sensitized animals produced larger responses (8-fold increases) at the same time point. A specific LTD4 receptor antagonist MK-571 (10 micrograms/eye) completely inhibited in vivo chemotactic responses to LTD4, and partially inhibited (54%) the responses to ovalbumin (observations subsequently confirmed by histological studies). As eosinophils may play an important role in allergic diseases, the results with MK-571 indicate that selective and potent LTD4 receptor antagonists may provide a novel therapy for allergic conjunctivitis, rhinitis and asthma.

Topics: Animals; Chemotaxis, Leukocyte; Conjunctiva; Eicosanoids; Eosinophils; Guinea Pigs; Histamine; Histocytochemistry; Indium Radioisotopes; Male; Ovalbumin; Platelet Activating Factor; Propionates; Quinolines; Receptors, Immunologic; Receptors, Leukotriene; SRS-A

The role of leukotriene D4 (LTD4) as a mediator of immediate hypersensitivity reactions in the guinea-pig conjunctiva was examined using a potent, second-generation LTD4 receptor antagonist, MK-571 (also known as L-660,711). The microvascular permeability changes in the guinea-pig conjunctiva following challenge with either LTD4 or antigen were measured through accumulation of intravenously administered 99mtechnetium-labeled albumin. Topical application of MK-571 (up to 2 h pretreatment) significantly inhibited the conjunctival responses to LTD4 (ED50 of 18-60 ng/eye) but not to histamine. The responses to a single topical antigen challenge in ovalbumin-sensitized guinea pigs were significantly inhibited (44%) by topical treatment with MK-571, in contrast to the lack of effect previously observed with prototypic antagonists. The inhibitory effects of MK-571 did not involve an action on conversion of [3H]LTC4 to LTD4 and LTE4. Following a second antigen challenge (24 h after the first), MK-571 inhibited the resultant permeability changes by 78%. Specific histamine H1 and H2 antagonists similarly inhibited the responses to the first and second challenges (63 and 74%, respectively). The present study suggests that LTD4 is involved in conjunctival hypersensitivity reactions and that potent LTD4 receptor antagonists may be of therapeutic value in the treatment of allergic conjunctivitis.

Topics: Animals; Conjunctiva; Guinea Pigs; Histamine; Hypersensitivity, Immediate; Leukotriene E4; Male; Ovalbumin; Propionates; Quinolines; Receptors, Immunologic; Receptors, Leukotriene; SRS-A