Inhalation of specific allergens usually results in increased airways resistance in asthmatic subjects. This reversible airway obstruction is associated with release of mast cell mediators and an inflammatory response in the airways. In the present studies, we used bronchoalveolar lavage (BAL) to directly assess local inflammatory changes occurring within the airways of atopic asthmatic subjects undergoing experimental bronchoprovocation (BPC), or during environmental, ie, “spontaneous” seasonal exposure. We also examined changes in local cell populations and evaluated lung eosinophils and mast cells morphologically for evidence of degranulation.
Seventeen subjects volunteered for these studies. Twelve had mild allergic asthma and five were asymptomatic normal individuals. All subjects gave informed consent to undergo BAL and aeroallergen BPC before entering this study which had been approved by the Human Subjects Use Committee of the University of Iowa. These studies also strictly conform to the guidelines set forth by the American College of Chest Physicians, American Academy of Allergy and Immunology – https://www.aaaai.org/home.aspx, American Thoracic Society, the National Heart, Lung and Blood Institute, and the National Institutes of Allergy and Infectious Diseases. In order to evaluate each individuals atopic status, a complete medical history and physical examination were performed, and a routine battery of allergen skin tests was applied intradermally to the triceps area of the upper arm. A wheal 5×5 mm or greater than diluent control was considered positive. Specific allergens used for BPC were titered on the forearm and observed at 30 minutes and six hours. Histamine and codeine positive control substances were also applied. The atopic asthmatic individuals gave a history of mild seasonal asthma, were skin reactive to the appropriate allergen, developed bronchoconstriction in response to inhaled specific allergen, and demonstrated a positive methacholine aerosol challenge (Table 1). Normal control subjects had no symptoms of respiratory allergy treated by Canadian Health&Care Mall, negative skin tests, a negative methacholine challenge, and no immediate family history for allergic disease.
Criteria for Asthmatic Subjects Undergoing BAL
In order to reduce the risk of triggering already hyperresponsive airways and to maintain rigorous control of those who would be challenged and lavaged, we established specific criteria for all asthmatic subjects (Table 2). Subjects were between the ages of 18 and 45, nonsmokers, and had only mildly symptomatic seasonal asthma. Immediately prior to BAL, they were required to have an FEV1.
A methacholine (Meth) aerosol challenge was employed to document hyperresponsive or normally responsive airways. Using established procedures, we employed the Johns Hopkins Dosimeter and concentrations of Meth from 0.075 to 25 mg/ml. Subjects were given five breaths of each concentration of Meth by taking slow deep breaths from functional residual capacity to total lung capacity, without breath-holding. A drop in FEVt by 20 percent (below diluent baseline) which persisted for at least five minutes was considered a positive response. The provocative dose producing a 20 percent decrease in FEVj (PDM [FEVJ) was calculated, and expressed as breath units (BU).
Allergen inhalation was performed in a similar manner, using increasing five-fold concentrations of allergens from 1 protein nitrogen unit (PNU)/ml to 10,000 PNU/ml. A 20 percent or greater drop in FEV1 which persisted ^5 minutes was considered positive, and the challenge was stopped. Those subjects who responded to allergen challenge were closely observed in the Clinical Research Center (CRC) of the University of Iowa for 24 hours. Pulmonary function tests were obtained (every 15 minutes for 60 minutes and then every hour from a total of 24 hours) using a forced expiratory maneuver with the Jones Pulmonor II (FVC, FEV^ and MEFR^ts). Normal individuals as control subjects were also challenged with allergen, observed in the CRC overnight, and pulmonary functions were similarly obtained.
Atopic symptomatic (in season) asthmatic subjects who were not taking any medications were challenged out of the appropriate historical season with an allergen to which they were clinically sensitive. Altemaria was used primarily, but cat dander extract was inhaled by one subject, short ragweed extract by another, and house dust by a third. Normal subject control subjects were challenged with 10,000 PNU/ml Altemaria. The PD* for the airway response following allergen challenge was calculated using previously described methods.
BAL Following BPC
For safety reasons, in most instances, we wished to lavage patients when the FEVt was near baseline value. For this reason, we chose time periods two to four hours or 24 hours after BPC for BAL (all asthmatic patients had late phase responses which occurred at 4 to 24 hours after inhalation of allergen). Each subject was prepared for BAL in a manner which is routine for all bronchoscopies, including bronchoalveolar lavage, at the University of Iowa. Atropine sulphate, 0.6 mg IM, and morphine sulphate, 8 mg IM, were given one half hour before the procedure. Xylocaine (4 percent) was aerosolized into the upper airways and applied topically to the pryriform sinuses to prevent coughing and to effect local anesthesia. Each individual also inhaled two puffs (0.65 mg/puff) of metaproterenol 15 minutes prior to bronchoscopy. All patients and control subjects were medicated identically. Bronchoscopy was performed using a fiberoptic bronchoscope; the tip of the bronchoscope was wedged into three different subsegmental bronchi in the left upper lobe, left lower lobe, and right lower lobe for lavage. Lavage in each site was performed by injecting five 20-ml aliquots of warmed (37°C) normal saline solution (total = 100 ml). Immediately after the injection of each aliquot, suction was applied and the fluid recovered in a sterile trap. The volume of each lavage specimen was measured and then immediately transported to the laboratory.