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

  • 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
  • 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. )
  • 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 protocol 1)
  • Absolute ethanol, 4°C
  • Enzyme immunoassay (EIA) kits for, e.g., LTC 4/D 4/E 4, PGD 2, 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 protocol 1)

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

  Materials
  • Female BALB/c mice, ≥16 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 protocol 5)
  • Supernatant from uninfected HEp‐2.2 cells (see protocol 5)
  • 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 protocol 2)

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 recipecomplete 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) H 2O/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% CO 2 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

Videos

Literature Cited

Literature Cited
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   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.
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   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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