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.
Each subject was lavaged once at baseline, “out of season,” then on another occasion at either two to four hours, or 24 hours after BPC. One asthmatic subject was lavaged at two hours and 24 hours after BPC, each on separate occasions. The details of the timing of these lavages are found in Table 1. Tliese lavages were separated by at least two weeks, and in most cases, four to six weeks or more. Five subjects were lavaged “in-season” when they were mildly symptomatic or potentially affected by a seasonal exposure (ie, during the (all mold season), and received no experimental bronchoprovocation. These patients were mildly symptomatic within a known allergy season. We postulated that such patients would be undergoing an active inflammatory response akin to a sustained late response.
Bronchoalveolar Lavage Specimens
The lavage fluid was immediately filtered through two layers of sterile gauze, and the cells were pelleted at 250 x g for five minutes. The lavage supemate was aliquoted and stored at — 70°C for further studies. The cells were washed with Hanks balanced buffered salt solution (HBSS; without Ca++ or Mg++) x2 and counted with a Coulter counter. The cells were then resuspended in RPMI 1640 medium with 5 percent fetal calf serum (FCS) to a concentration of 1 x 107 cells/ml.
Bronchoalveolar cells were evaluated using cytocentrifuge preparations stained with Wnght-Giesma. Two hundred cells were counted and the percentage of cells which were macrophages, lymphocytes, neutrophils, mast cells, and eosinophils was determined. For each patient, the data from the three lavage sites were averaged and expressed as cells per 100 ml lavage; thus, a total of600 cells were counted. Mast cells were easily differentiated from other mono-cytoid cells by their purple-staining granules, size, and eccentric nuclei.
Pelleted lavage cells (1-2 x 106 cells) were washed x 1 with HBSS and then resuspended in 5 ml of HBSS, added to 50 percent modified colloidal silica (Percoll) and centrifuged at 800 x g for 30 minutes as described elsewhere. The cell pellet was immediately fixed with 2.5 percent glutaraldehyde. Pelleted cells were postfixed with 2 percent osmium tetroxide and embedded in Spurr s plastic before thin-sectioning with a diamond knife using a MT-2B ultramicrotome. Thin sections (600 to 900A°) were mounted on uncoated copper grids and stained with uranyl acetate and lead citrate using standard methods. Thin sections were examined using an electron microscope.
Data were evaluated using a double tail Students f-test. Where appropriate, paired samples were compared; p<0.05 was considered significant.
Table 1—Clinical Characteristics of Atopic Asthmatic and ‘Normal Subjects
|Time After Aerosol Challenge|
|Baseline||<4 Hrs||24 hrs||Seasonal|
|Normal subjects (5)||3-F2-M||21-35||AIT||NR||X||xt|
Table 2—Criteria for Bronchoalveolar Lavage Following Early and Late Phase Asthmatic Responses
|—Age: 18 to 45|
|—Mildly symptomatic of seasonal allergic asthma|
|—Positive skin test|
|—Positive methacholine challenge|
|—FEV! before lavage is ^60 percent of predicted, and not less than 2 L|