Models of Pulmonary Disease: Acute and Chronic Allergic Asthma in the Monkey and Acute and Chronic Viral Pulmonitis in the Mouse

Craig D. Wegner1, Thomas Shaughnessy1, Donna Mussatto1

1 Abbott Laboratories, Abott Park, Illinois
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 5.2
DOI:  10.1002/0471141755.ph0502s00
Online Posting Date:  May, 2001
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Abstract

Three models/protocols designed to mimic an inflammation process characteristic of a specific lung disease are described in this unit. Included are a single allergen inhalation in monkeys to model acute asthmatic episodes, repeated allergen inhalations in monkeys to model chronic asthma, and a lower-respiratory viral infection in mice to model acute and chronic viral alveolitis and bronchitis. In each case, alterations in lung functions believed to correlate with symptoms of the respective disease are measured along with pulmonary inflammation.

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol 1: Inhaled Allergen–Induced Acute Responses in Primates
  • Support Protocol 1: Processing of Bronchoalveolar Lavage (BAL) Samples
  • Basic Protocol 2: Repeated Inhaled Allergen–Induced Airway Hyperresponsiveness and Eosinophilia in Primates as a Model of Chronic Asthma
  • Basic Protocol 3: Lung Inflammation and Dysfunction Associated with Respiratory Syncytial Virus (RSV) Infection in Mice
  • Support Protocol 2: Preparation of High-Titer Respiratory Syncytial Virus (RSV) Stocks
  • Reagents and Solutions
  • Commentary
  • Bibliography
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Inhaled Allergen–Induced Acute Responses in Primates

 Materials
  • Adult male cynomolgus monkeys (Macaca fascicularis), 4 to 8 kg, with naturally occurring skin hypersensitivity to Ascaris suum extract (Charles River Labs)
  • Ketamine hydrochloride (Ketaset; Fort Dodge Animal Health)
  • Xylazine (Rompun; Bayer Corporation Agricultural Division)
  • Ascaris suum extract (Greer Labs)
  • Phosphate-buffered saline (PBS; see recipe)
  • Bicarbonate-buffered saline (BBS; see recipe)
  • Squeeze-back monkey cage (Hazelton Research)
  • Monkey support chair (specially designed for support around neck and seat; Fig. 5.2.1)
     FigureFigure 5.2.1 Specially designed support chair for upright-seated positioning of an anesthetized monkey.
  • Cuffed endotracheal tube (4.5-, 5.0-, 5.5-mm i.d; Baxter)
  • Small-dog laryngoscope and jaw spreader (Henry Schein)
  • Large-volume (500-ml) syringe (Harvard Apparatus)
  • Compressed-air micronebulizer (Bird Products model 8158)
  • Positive-pressure ventilator (Bird Products model Mark 7A)
  • Instrumentation for measuring respiratory function including:
  •     Differential pressure transducers
  •     Appropriate tubing and connectors
  •     Pneumotachograph (Fleisch, model 0)
  •     Software and hardware for measurement of pulmonary mechanics, e.g.: Biowindows/Bioreports (Modular Instruments) for standard resistance and compliance measurements; forced oscillations setup (Pulmetrics) for oscillatory impedance measurements; or instrumentation from Buxco for either technique
  • V-shaped, heated surgical table (optional; Baxter)
  • Pediatric fiberoptic bronchoscope with lavage/biopsy channel (Olympus)
  • 20-ml syringe

Support Protocol 1: Processing of Bronchoalveolar Lavage (BAL) Samples

 Materials
  • BAL fluid sample (see Basic Protocol 1)
  • Absolute ethanol, 4°C
  • Enzyme immunoassay (EIA) kits for, e.g., LTC4/D4/E4, PGD2, human IL-1, GM-CSF, IL-6, and IL-8
  • Leukocyte counter (e.g., Coulter model Z1)
  • Isoton buffer (Coulter)
  • Cytocentrifuge (e.g., Cytospin model 3; Shandon/Lipshaw)
  • Refrigerated centrifuge
  • Speedvac evaporator

Basic Protocol 2: Repeated Inhaled Allergen–Induced Airway Hyperresponsiveness and Eosinophilia in Primates as a Model of Chronic Asthma

 Materials
  • Adult male cynomolgus monkeys (Macaca fascicularis), 4 to 8 kg, with naturally occurring hypersensitivity to Ascaris suum extract, which have received Ascaris inhalation challenges intermittently for several months (preferably 1 to 2 years) (Charles River Labs)
  • Methacholine (Sigma)
  • Additional reagents and equipment for inducing and measuring acute responses to inhaled allergen in primates and performing BAL (see Basic Protocol 1)

Basic Protocol 3: Lung Inflammation and Dysfunction Associated with Respiratory Syncytial Virus (RSV) Infection in Mice

 Materials
  • Female BALB/c mice, ³16 weeks old (preferably ~32 weeks old), e.g., retired breeders (Charles River Labs)
  • Sevoflurane (Abbott Labs)
  • Supernatant from HEp-2.2 cells containing 1 × 107 pfu/ml respiratory syncytial virus (RSV), A2 (long group A) strain (see Support Protocol 2)
  • Supernatant from uninfected HEp-2.2 cells (see Support Protocol 2)
  • Sodium pentobarbital (Abbott Labs)
  • Gas mixture (in cylinder): ~0.5% carbon monoxide, 0.5% neon, 20% oxygen, and 79% nitrogen (Matheson Gas Products)
  • Methacholine (Sigma)
  • Phosphate-buffered saline (PBS; see recipe), pH 7.4
  • Phosphate-buffered saline (PBS; see recipe), pH 7.4, containing 0.1% EDTA
  • Lung homogenate medium (see recipe)
  • 18-G Teflon catheters with luer plugs (Baxter), cut to a 45° angle at one end
  • 1-ml and 3-ml syringes
  • 3-way stopcock
  • Analytical gas chromatograph (e.g., Carle AGC series 100, model 01111)
  • Instrumentation for measuring respiratory function including:
  •     Differential pressure transducer(s)
  •     Appropriate tubing and connectors
  •     Software and hardware for measurement of pulmonary mechanics, e.g.: Biowindows/Bioreports (Modular Instruments); LabWindows from Buxco for standard resistance/compliance measurements or free-roaming measurements of PenH/Epause; or forced oscillations setup (Pulmetrics) for oscillatory impedance measurements
  • Ultrasonic nebulizer (e.g., DeVilbiss Aerosonic, model 5000D)
  • Small-animal ventilator (Harvard Apparatus)
  • Tissue homogenizer
  • Refrigerated centrifuge
  • Additional reagents and equipment for processing broncheoalveolar lavage (BAL) samples (see Support Protocol 1)

Support Protocol 2: Preparation of High-Titer Respiratory Syncytial Virus (RSV) Stocks

 Materials
  • HEp-2.2 cells (ATCC #CCL 23)
  • Complete MEM medium/10% FBS (see recipe) and serum-free MEM
  • Respiratory syncytial virus (RSV), A2 (long group A) strain (ATCC #VR 1302)
  • 0.75% (w/v) methylcellulose in complete MEM medium/10% FBS (prepare 1 day in advance; methylcellulose requires time to go into solution)
  • Dulbecco's PBS (Life Technologies) containing Mg2+ and Ca2+
  • 10% (v/v) formalin
  • 1% (w/v) crystal violet in 50% (v/v) H2O/methanol
  • T-75 tissue culture flasks
  • Cell scrapers
  • Inverted microscope
  • 50-ml conical centrifuge tubes
  • Sonicator
  • Tabletop centrifuge, 4°C
  • 24- or 96-well tissue culture plates

NOTE: All culture incubations are performed in a humidified 37°C, 5% CO2 incubator unless otherwise specified.

NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.
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Figures

  •  FigureFigure 5.2.1 Specially designed support chair for upright-seated positioning of an anesthetized monkey.
    View Image
  •  FigureFigure 5.2.2 Preparation of mouse for measurement of diffusion capacity of the lungs with respect to carbon monoxide.(A) A three-way stopcock is connected to the trachea cannula, an empty 1-ml syringe, and a 1-ml syringe filled with 0.5% CO/0.5% Ne/20% O2/79% N2 gas mixture. (B) The gas mixture is injected into the lungs and held for 10 sec. (C) Immediately at the end of 10 sec, a 0.5-ml dead-space (airway) gas sample is withdrawn into one of the syringes. This is followed immediately by (D) in which a 0.5-ml “alveolar” gas sample is withdrawn into the other syringe. The stopcock is then closed and the syringes and stopcock disconnected from the animal and connected to a gas chromatograph for analysis of the alveolar gas sample.
    View Image
  •  FigureFigure 5.2.3 Preparation of mouse for collection of lung lavage and tissue homogenate samples. (A) A midline abdominal incision is made to the tracheostomized animal to expose the diaphragm. (B) The diaphragm is then punctured, whereupon the lungs collapse, and the chest plate is removed by cutting along the deflection point of the ribs on both sides of the sternum. The lungs are then visible for checking inflation during lung lavage and/or removal for the determination of viral titers.
    View Image
  •  FigureFigure 5.2.4 Time course of (A) leukocyte infiltration; (B) Increase in viral (RSV) titers; and (C) body weight loss induced by RSV-infection in mice. Bars represent the mean ± standard error. Asterisks signify a significant difference from baseline and medium-inoculated control animals (p <0.05 by Student's t-test).
    View Image

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Literature Cited

 Literature Cited
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    Boushey, H.A., Holtzman, M.J., Sheller, J.R., and Nadel, J.A. 1980. State of the art: Bronchial hyperreactivity. Am. Rev. Respir. Dis. 121:389-413.
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    Graham, B.S., Bunton, L.A., Wright, P.F., and Karzon, D.T. 1991. Role of T lymphocyte subsets in the pathogenesis of primary infection and rechallenge with respiratory syncytial virus in mice. J. Clin. Invest. 88:1026-1033.
    Gundel, R.H., Gerritsen, M.E., Gleich, G.J., and Wegner, C.D. 1990. Repeated antigen inhalation results in a prolonged airway eosinophilia and airway hyperresponsiveness in primates. J. Appl. Physiol. 68:779-786.
    Gundel, R.H., Wegner, C.D., Torcellini, C.A., Clarke, C.C., Haynes, N., Rothlein, R., Smith, C.W., and Letts, L.G. 1991a. Endothelial leukocyte adhesion molecule-1 mediates antigen-induced acute airway inflammation and late-phase airway obstruction in monkeys. J. Clin. Invest. 88:1407-1411.
    Gundel, R.H., Kinkade, P., Torcellini, C.A., Clarke, C.C., Watrous, J., Desai, S., Homon, C.A., Farina, P.R., and Wegner, C.D. 1991b. Antigen-induced mediator release in primates. Am. Rev. Respir. Dis. 144:76-82.
    Gundel, R.H., Wegner, C.D., Heuer, H.O., and Letts, L.G. 1992a. A PAF receptor antagonist inhibits acute airway inflammation and late-phase responses but not chronic airway inflammation and hyperresponsiveness in a primate model of asthma. Mediat. Inflamm. 1:379-384.
    Gundel, R.H., Wegner, C.D., and Letts, L.G. 1992b. Antigen-induced acute and late-phase responses in primates. Am. Rev. Respir. Dis. 146:369-373.
    Kim, H.W., Canchola, J.G., Brandy, C.D., Pyles, G., Chanock, R.M., Jensin, K., and Parrott, R.H. 1969. Respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine. Am. J. Epidemiol. 89:422-434.
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    Pullan, C.R. and Hey, E.N. 1982. Wheezing, asthma and pulmonary dysfunction 10 years after infection with respiratory syncytial virus in infancy. Br. Med. J. 284:1665-1669.
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    Robinson, J.P., Darzynkiewicz, Z., Dean, P.N., Orfao, A., Rabinovitch, P.S., Stewart, C.C., Tanke, H.J., and Wheeless, L.L. (eds.) 1997. Current Protocols in Cytometry. John Wiley & Sons, New York.
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    Souza, D.J., Gundel, R.H., Barton, R.W., Stearns, C.D., Torcellini, C.A., Miner, E.J., Letts, L.G., and Wegner, C.D. 1993. Changes in T-lymphocyte subsets and activation following chronic antigen inhalations in monkeys. Chest 103:S132-S133.
    Stott, E.J., Ball, L.A., Anderson, K., Young, K.K.Y., King, A.M.Q., and Wertz, G.W. 1987. Immune and histopathological responses in animals vaccinated with recombinant vaccinia viruses that express individual genes of human respiratory syncytial virus. J. Virol. 61:3855-3861.
    Strober, W. 1997. Wright-Giemsa and nonspecific esterase staining of cells. In Current Protocols in Immunology (J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, and W. Strober, eds.) pp. A.3C.1-A.3C.3. John Wiley & Sons, New York.
    Townley, R.G., Ryo, U.Y., Kolotkin, B.M., and Kang, B. 1975. Bronchial sensitivity to methacholine in current and former asthmatics and allergic rhinitis patients and control subjects. J. Allergy Clin. Immunol. 56:429-442.
    Turner, C.R., Andresen, C.J., Smith, W.B., and Watson, J.W. 1994. Effects of Rolipram on responses to acute and chronic antigen exposure in monkeys. Am. J. Respir. Crit. Care Med. 149:1153-1159.
    Wegner, C.D. 1994. Lung inflammation. In Adhesion Molecules (C.D.Wegner, ed.) pp. 191-208. Academic Press, London.
    Wegner, C.D. 1995. Chronic models of airway hyperresponsiveness Eur. Respir. Rev. 5/29:218-223.
    Wegner, C.D., Gundel, R.H., Reilly, P., Haynes, N., Letts, L.G., and Rothlein, R. 1990. Intercellular adhesion molecule-1 (ICAM-1) in the pathogenesis of asthma. Science 247:456-459.
    Wegner, C.D., Torcellini, C.A., Clarke, C.C., Letts, L.G., and Gundel, R.H. 1991. Effects of single and multiple inhalations of antigen on airway responsiveness in monkeys. J. Allergy Clin. Immunol. 87:835-841.
    Wegner, C.D., Gundel, R.H., Abraham, W.M., Schulman, E.S., Kontny, M.J., Lazer, E.S., Homon, C.A., Graham, A.G., Torcellini, C.A., Clarke, C.C., Jager, P., Wolyniec, W.W., Letts, L.G., and Farina, P.R. 1993. The role of 5-lipoxygenase products in preclinical models of asthma. J. Allergy Clin. Immunol. 91:917-929.
 Key References
    Geba, G.P., Wegner, C.D., Wolyniec, W.W., Li, Y., and Askenase, P.W. 1997. Nonatopic asthma: In vivo airway hyperreactivity adoptively transferred to naive mice by THY-1+ and B220+ antigen-specific cells that lack surface expression of CD3. J. Clin. Invest. 100:629-638.

Description of measurement of pulmonary function including airway responsiveness in mice as well as techniques for analysis and transfer of immune cells.

    Gundel et al., 1991b. See above.

Measurement of allergen-induced acute mediator release in primates.

    Gundel et al., 1992b. See above.

Characteristics and measurement of late-phase bronchoobstruction and leukocyte infiltrate in monkeys.

    Turner et al., 1994. See above.

Measurement of allergen-induced late-phase cytokines/chemokines.

    Wegner, et al., 1991. See above.

Description of induction of airway hyperresponsiveness and eosinophilia in monkeys by alternate allergen inhalations.

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