Pulmonary inflammation with eosinophil (EOs) infiltration is a prominent feature of allergic respiratory diseases such as asthma. In order to study the cellular response during the disease development, an animal model of IgE-mediated pulmonary inflammation with characteristic eosinophilia is needed. We developed a method for inducing severe pulmonary eosinophilia in the mouse and also studied the numbers of EOs in blood and bone marrow and the response to corticosteroid treatment. Animals were sensitized with alum-precipitated ovalbumin (OVA) and challenged with aerosolized OVA 12 days later when serum IgE levels were significantly elevated. Four to eight hours after challenge there were moderate increases in the number of EOs in the bone marrow and peripheral blood, but only a few EOs were observed in the lung tissue and in bronchoalveolar lavage (BAL) fluid. Twenty-four hours after challenge, there was a marked reduction of EOs in bone marrow, while the number of EOs peaked in the perivascular and peribronchial regions of the lung. Forty-eight hours after challenge, the highest number of EOs was found in the BAL fluid, making up > 80% of all cells in that compartment. The high levels of EOs in the lung tissue and BAL fluid lasted for 2-3 days and was followed by a more moderate but persistent eosinophilia for another 10 days. Nonsensitized animals showed no significant changes in the number of EOs in BAL fluid, lungs, blood or bone marrow. Histopathological evaluation also revealed epithelial damage, excessive mucus in the lumen and edema in the submucosa of the airways. The pulmonary eosinophilia and decrease in bone marrow EOs induced by OVA challenge responded well to treatment with several standard corticosteroids. The rank order of steroid potency for inhibition of pulmonary eosinophilia was betamethasone > prednisolone > hydrocortisone. Because mice are extremely useful for immunological studies, this model can be invaluable to study the effects of cytokines on pulmonary inflammation.

To investigate the role of interleukin-5 (IL-5) on airway hyperreactivity and pulmonary inflammation in nonhuman primate airways, the effect of a neutralizing monoclonal antibody to murine IL-5 (TRFK-5) was investigated in a cynomolgus monkey model of allergic asthma. Anesthetized Ascaris-sensitive monkeys underwent bronchoalveolar lavage (BAL) to assess the granulocyte content of this fluid before and 24 h after aerosolized Ascaris suum extract inhalation. Airway reactivity was assessed by the concentration of inhaled histamine required to produce a 40% reduction in dynamic lung compliance (Cdyn40). Exposure to A. suum extract produced an increase in airway reactivity (Cdyn40 = 0.065 +/- 0.024% before Ascaris; Cdyn40 = 0.014 +/- 0.004% after Ascaris) and an inflammatory reaction in the airways characterized by an increase in BAL eosinophils (0.05 +/- 0.03 x 10(3) cells/ml before Ascaris; 176 +/- 76 x 10(3) cells/ml after Ascaris) and neutrophils (3 +/- 1 x 10(3) cells/ml before Ascaris; 406 +/- 211 x 10(3) cells/ml after Ascaris). In contrast, only small nonsignificant changes in airway reactivity and granulocyte influx into the BAL occurred after aerosolized saline as a sham challenge. When the monkeys were treated 1 h before Ascaris challenge with the TRFK-5 antibody (0.3 mg/kg, intravenously), there was no increase in airway reactivity after Ascaris challenge (Cdyn40 = 0.032 +/- 0.016% before Ascaris; Cdyn40 = 0.217 +/- 0.196% after Ascaris) and there were only small increases in the number of eosinophils and neutrophils in the BAL after Ascaris challenge. The inhibition of this pulmonary eosinophilia and bronchial hyperresponsiveness by TRFK-5 was seen for up to 3 mo after treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

To investigate the role of IL-5 in airway hyperreactivity and pulmonary eosinophilia, we used a model of allergic asthma in guinea pigs and a neutralizing monoclonal antibody (TRFK-5) directed against murine IL-5. Sensitized guinea pigs were challenged with 1% ovalbumin (OVA) aerosol and assessed for airway eosinophilia (by bronchoalveolar lavage [BAL] and histologic evaluation of airway tissue) and bronchoconstrictor responsiveness to substance P (SP) (as RL100 and Cdyn40) 24 h later. OVA challenge of sensitized animals caused a significant increase in airway responsiveness to SP, with a 4.9-fold decrease in RL100 and a 4.7-fold decrease in Cdyn40. Accompanying this increased sensitivity to SP was a 9-fold increase in eosinophils recovered in BAL and a 4- to 5-fold increase in eosinophils in intrapulmonary bronchial tissue. Intraperitoneal treatment with 10 mg/kg of the IL-5 antibody 2 h before OVA challenge blocked BAL and lung tissue increases in eosinophils but had no effect on the development of airway sensitivity to SP. In contrast, similar treatment with 30 mg/kg of this antibody blocked OVA-induced increased sensitivity to SP as well as BAL and lung tissue eosinophilia. These data suggest a critical and possibly independent role for IL-5 in allergic airway hyperresponsiveness and the accumulation of eosinophils within the lung of the guinea pig.

Interleukin-5 (IL-5) is important in the control of differentiation, migration, and activation of eosinophils. In order to study the role of IL-5 in the development of eosinophilic inflammation of the airways, we have used a monoclonal antibody to murine IL-5 (TRFK-5) in a murine model of allergic pulmonary inflammation. B6D2F1 mice were sensitized with alum-precipitated ovalbumin and were challenged with aerosolized ovalbumin on day 12 after sensitization. Samples of bronchoalveolar lavage (BAL) fluid, lung tissue, blood, and bone marrow aspirate were collected at different times after ovalbumin challenge. Twenty-four hours after challenge there were significant increases in the number of eosinophils in the BAL fluid, lung tissue, and blood while bone marrow eosinophils were decreased. Treatment of sensitized mice with TRFK-5 (0.01-1 mg/kg, i.p.) 2 h before ovalbumin challenge reduced the numbers of eosinophils in the BAL fluid and lung tissue and prevented the decrease in bone marrow eosinophils in a dose-dependent fashion. The number of eosinophils in the BAL fluid, peribronchial and alveolar regions of the lung was also reduced when TRFK-5 (2 mg/kg, i.p.) was given up to 5 d after ovalbumin challenge. Furthermore, there was no evidence of increased epithelial damage, edema, or the presence of mucus that could have resulted from eosinophil apoptosis and release of toxic proteins after neutralization of IL-5. These results demonstrate an important role for IL-5 in the development of eosinophilic inflammation of the airways and for the migration of eosinophils from the bone marrow into blood in response to antigen challenge.(ABSTRACT TRUNCATED AT 250 WORDS)

Mast cells are important effector cells in IgE-mediated acute allergic reactions. Mast cells also produce cytokines such as interleukin (IL)-3, IL-4, IL-5, tumor necrosis factor (TNF), and granulocyte-macrophage colony-stimulating factor (GM-CSF) that regulate the function of eosinophils and the development of a late-phase inflammatory response to antigen challenge. To evaluate the role of mast cells on the development of IgE-mediated allergic pulmonary eosinophilia in vivo, we compared the eosinophil infiltration into lungs of mast cell deficient mice (WBB6F1/J-W/Wv) with their congenic normal littermates (W/W+). Mice were sensitized with alum-precipitated ovalbumin and challenged with aerosolized ovalbumin on day 12 after sensitization. Bronchoalveolar lavage (BAL) fluid, lung tissue biopsies, and blood samples were collected after ovalbumin challenge. Eosinophil numbers in the BAL and lung tissue, lung eosinophil peroxidase (EPO) activity and serum levels of IgE and IgG1 were measured. In sensitized W/W+ mice, there were increased numbers of eosinophils in the BAL fluid and lung tissue, and EPO levels were increased after ovalbumin challenge. Ovalbumin challenge of sensitized mast-cell-deficient mice produced fewer numbers of eosinophils in the BAL fluid and lungs, and EPO levels were also reduced compared with their challenged congenic littermates. On the other hand, levels of serum IgE and IgG1 were not different between W/Wv mice and their congenic littermates.(ABSTRACT TRUNCATED AT 250 WORDS)

Nitric oxide (NO) is an important mediator of inflammatory reactions and may contribute to the lung inflammation in allergic pulmonary diseases. To assess the role of NO in pulmonary inflammation, we studied the effect of four nitric oxide synthase (NOS) inhibitors, N-nitro-L-arginine methyl ester (L-NAME), aminoguanidine, N(G)-monomethyl-L-arginine (NMMA) and L-N6-(1-Iminoethyl) lysine (L-NIL), on the influx of eosinophils into the bronchoalveolar lavage (BAL) fluid and lung tissue of antigen-challenged allergic mice. We also analyzed lung tissues for the presence of steady state mRNA for inducible nitric oxide synthase (iNOS) and iNOS protein. Furthermore, BAL fluid and serum were analyzed for their nitrite content. B6D2F1/J mice were sensitized to ovalbumin (OVA) and challenged with aerosolized OVA. The NOS inhibitors were given 0.5 h before and 4 h after the antigen challenge. OVA challenge induced a marked eosinophilia in the BAL fluid and lung tissue 24 h after challenge. The OVA-induced pulmonary eosinophilia was significantly reduced by L-NAME (10 and 50 mg/kg, intraperitoneally [i.p.]). The inactive isomer, D-NAME (50 mg/kg, i.p.) had no effect. When mice were treated with L-NAME (20 mg/kg, i.p.) and an excess of NOS substrate, L-arginine (200 mg/kg, i.p.), the OVA-induced pulmonary eosinophilia was restored. Treatment with aminoguanidine (0.4-50 mg/kg, i.p.) also reduced the pulmonary eosinophilia. Treatment with NMMA (2-50 mg/kg, i.p.) partially reduced the eosinophilia, but L-NIL (10-50 mg/kg, i.p.), a selective iNOS inhibitor, had no effect. L-NAME had no effect on the reduction of eosinophils in the bone marrow following OVA challenge to sensitized mice. OVA challenge to sensitized mice had no effect on iNOS protein expression or iNOS mRNA in the lungs or on the levels of nitrite in the BAL fluid. These results suggest that NO is involved in the development of pulmonary eosinophilia in allergic mice. The NO contributing to the eosinophilia is not generated through the activity of iNOS nor does NO contribute to the efflux of eosinophils from the bone marrow in response to antigen challenge. It is speculated that after antigen challenge, the localized production of NO, possibly from pulmonary vascular endothelial cells, is involved in the extravasation of eosinophils from the circulation into the lung tissue.

1 The GABA-B receptor agonists baclofen and 3-aminopropylphosphinic acid (3-APPi) have antitussive activity in the cat and guinea pig. The purpose of this study was to investigate the sites of action of these GABA-B receptor agonists to inhibit the cough reflex. 2 Single intracerebroventricular (i.c.v.) cannulas were placed in the lateral ventricles of anaesthetized guinea pigs. Approximately 1 week later, the animals were exposed to aerosols of capsaicin (0.3 mM) to elicit coughing. Coughs were detected with a microphone and counted. 3 Cough was produced in anaesthetized cats by mechanical stimulation of the intrathoracic trachea and was recorded from electromyograms of respiratory muscle activity. Cannulas were placed for intravenous (i.v.) or, in separate groups of animals, intravertebral arterial (i.a.) administration of baclofen, 3-APPi, the centrally active antitussive drug codeine or the peripherally active antitussive drug BW443c. Dose-response relationships for i.v. and i.a. administration of each drug were generated to determine a ratio of i.v. ED" to i.a. ED50, known as the effective dose ratio (EDR). The EDR will be 20 or greater for a centrally acting drug. 4 In the guinea pig, baclofen (3 mg kg-', s.c.) and 3-APPi (10 mg kg-', s.c.) inhibited capsaicininduced cough by 50% and 35% respectively. The antitussive activity of baclofen was completely blocked by i.c.v. administration of the GABA-B receptor antagonist CGP 35348 (10 jig). Conversely, the antitussive effect of 3-APPi was unaffected by i.c.v. CGP 35348. However, systemic administration of CGP 35348 (30 mg kg-', s.c.) completely blocked the antitussive activity of 3-APPi (10 mg kg-', s.c.). In separate experiments baclofen alone (1 gpg, i.c.v.) inhibited capsaicin-induced cough by 78%. 3-APPi (10 and 100 gg, i.c.v.) had no effect on capsaicin-induced cough in the guinea pig. 5In the cat, potencies (ED50) of the standards and GABA-B agonists by the i.v. route were: codeine (0.34 mg kg-'), BW443C (0.17 mg kg-'), baclofen (0.63 mg kg-') and 3-APPi (2.3 mg kg-'). Potencies of these drugs by the i.a. route were: codeine, 0.013 mg kg-'; BW443C, 0.06mg kg-'; baclofen, 0.016mg kg-'; and 3-APPi, 0.87 mg kg'. The EDRs for each drug were: codeine, 26; BW443C, 3; baclofen, 39; and 3-APPi, 3. 6 We conclude that in both the cat and guinea pig baclofen inhibits cough by a central site of action, while 3-APPi inhibits cough by a peripheral site of action.

Based on its involvement in eosinophil biology, interleukin 5 (IL-5) may play a role in the pulmonary eosinophilia associated with allergic reactions. We have examined that hypothesis using a neutralizing antibody to IL-5 in ovalbumin-sensitized guinea pigs challenged with aerosolized antigen. The extent of eosinophilia has been quantitated in bronchoalveolar lavage (BAL) and by histologic evaluation of lung tissue sections. Acute intraperitoneal administration of a rat IgG, monoclonal antibody to murine IL-5 derived from TRFK-5 cells prevented lung and BAL eosinophilia in a dose-dependent fashion at and above 10 micrograms per guinea pig. Treatment with either an experimentally irrelevant, isotype-matched antibody from GL113 cells or with heat-denatured IL-5 antibody was without effect. These studies demonstrate the importance of IL-5 to pulmonary eosinophilia in challenged, allergic guinea pigs.