Bronchial Neutrophilia in Patients with Noninfectious Status Asthmaticus

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1 Bronchial Neutrophilia in Patients with Noninfectious Status Asthmaticus CATHERINE LAMBLIN, PHILIPPE GOSSET, ISABELLE TILLIE-LEBLOND, FABIENNE SAULNIER, CHARLES-HUGO MARQUETTE, B. WALLAERT, and A. B. TONNEL Clinique des Maladies Respiratoires, Hôpital A. Calmette, CHRU, Lille; INSERM U416, Institut Pasteur, BP 245, Lille Cedex; and Service d Urgences Respiratoires et de Réanimation Médicale, Hôpital A. Calmette, CHRU, Lille, France Cellular events that occur in status asthmaticus (SA) remain poorly investigated. Autopsy studies frequently emphasized about the presence of eosinophils in bronchial airway wall, whereas recent studies reported increased number of neutrophils in patients dying of sudden-onset fatal asthma. Mucus plugs occluding the bronchial lumen are almost constant features during SA. Bronchial lavage (BL) may be useful to remove mucus plugs in cases of atelectasis and/or refractory SA. We investigated the contribution of different cell types and cellular mediators (neutrophil elastase, eosinophil cationic protein [ECP], histamine, interleukin-8 [IL-8]) to the pathogenesis of SA. We studied 16 BL from eight patients undergoing mechanical ventilation (MV) for SA (time interval from onset of MV Day 0 to Day 11), four BL from patients undergoing MV without preexisting respiratory disease (V), 11 BL from patients with stable asthma (A) and eight BL from healthy controls (C). SA exhibited higher number and percentage of neutrophils ( %) than V ( ) (p 0.05), A ( ) and C ( ) (p ), and higher number of eosinophils than V, A, and C (p 0.01). Neutrophil elastase, ECP, and IL-8 levels were dramatically increased in SA. Histamine was higher in SA than in C and V (p 0.05). Bronchial neutrophilia was not related to concomitant bacterial infection as bacteriological cultures were positive in only three BL. Eosinophils, mast cells and histamine were higher in BL performed within the first 48 h of MV (p 0.05) than in BL performed later on. Our results indicate that bronchial inflammation in SA differs from bronchial inflammation in mild asthma. Persistent bronchial neutrophilia is associated with increased eosinophils and mast cells in the early phase of SA. Neutrophils may result in tissue damage and participate to the shedding of the epithelium in SA. Lamblin C, Gosset P, Tillie-Leblond I, Saulnier F, Marquette C-H, Wallaert B, Tonnel AB. Bronchial neutrophilia in patients with noninfectious status asthmaticus. AM J RESPIR CRIT CARE MED 1998;157: (Received in original form February 24, 1997 and in revised form August 28, 1997) Correspondence and requests for reprints should be addressed to Pr. A. B. Tonnel, M.D., Clinique des Maladies Respiratoires, Hôpital A. Calmette, Boulevard du Professeur Leclercq, Lille cedex, France. andre.tonnel@pasteur-lille.fr Am J Respir Crit Care Med Vol 157. pp , 1998 Asthma is a disease characterized by reversible airflow obstruction. Airways inflammation consisting with airways infiltration by eosinophils and activated T-lymphocytes is now recognized as central to the pathophysiology of asthma, in conjunction with smooth muscle bronchoconstriction and intraluminal mucus accumulation (1, 2). In some cases, patients with asthma develop life-threatening asthma attack referred to as status asthmaticus (SA). Postmortem studies of fatal asthma have provided knowledges of airway structural changes occurring during SA. These studies repeatedly demonstrated extensive mucus plugs in the airway lumen, shedding of the bronchial epithelium, airway smooth muscle hypertrophy, mucus gland hyperplasia, thickening of the basement membrane, and infiltration of the airway wall with inflammatory cells (3 9). These pathological changes, although more severe, reassembled those described in mild asthma. Nonetheless, the respective role of inflammatory cells and the dynamic cellular events occurring during the course of SA remain poorly investigated. Most of postmortem studies emphasized about the presence of eosinophils in the bronchial wall or in the mucous plugs filling the bronchi (3, 4, 8). Controversy exists regarding the critical role of the neutrophil as an effector cell in SA. Recent autopsy studies have shown a relationship between the time interval from the onset of asthma attack to fatal outcome and histological findings in terms of airway infiltration. In these studies, neutrophils appear to be the predominant cells in case of sudden-onset fatal asthma, whereas eosinophils are preferentially increased in cases of progressive-onset fatal asthma (6, 7, 9). In addition, Fahy and coworkers have demonstrated a prominent neutrophilic inflammation mediated by interleukin-8 (IL-8) in sputum from asthmatic patients admitted for acute severe asthma (10). Diffuse bronchial obstruction with viscid secretions is almost a constant feature in SA. As a consequence, well-characterized atelectasis and/or acute respiratory failure may ensue despite optimal medical treatment and ventilatory support. In such circumstances of refractory SA, bronchial lavage (BL)

2 Lamblin, Gosset, Tillie-Leblond, et al.: Bronchial Neutrophilia in Status Asthmaticus 395 may be helpful to remove the viscid plugs (11). To our knowledge, no data regarding cellular components of BL during SA are available. In an attempt to elucidate cell effectors and mediators playing a role in the setting of SA, we studied BL samples from patients with SA obtained at different time intervals from the onset of MV. However it must be stressed that several factors are prone to influence the cellular events that occur within the bronchi of patients undergoing MV for SA. Indeed, the medications administered in case of SA, especially corticosteroids, as well as ventilatory support, whom effects on bronchial cellular compartments are unknown, and/or a concomitant respiratory tract infection may modify the bronchial inflammatory response. Thus, we studied BL from patients undergoing MV without preexisting respiratory disease to evaluate the effects of MV on bronchial cellular events. We also compared BL from patients with SA with BL from mild asthmatics and BL from healthy volunteers in order to assess whether the inflammation seen in SA was qualitatively or quantitatively different from that observed in stable asthma. All BL were systematically assessed for bacteriological examination and were analyzed for total and differential cell counts, and for concentrations of neutrophil elastase, eosinophil cationic protein (ECP), histamine and interleukin-8 (IL-8). METHODS Patients Eight patients with status asthmaticus (SA) were included in this study. All had previous history of asthma. They were admitted in ICU between January 1995 and May 1996 with respiratory failure due to severe asthma attack requiring the onset of mechanical ventilation (MV). Particular attention was paid to the probable trigger of the asthma attack (head or chest cold, exposure to aeroallergen and/or nonspecific irritants, exercise, psychological conflicts). In addition, the time elapsed between the onset of the first symptoms and respiratory failure was estimated. Hospital treatment was conducted according to the current ATS recommendations (11) and consisted with corticosteroids (methylprednisolone 2 mg/kg/d), high dosages of bronchodilators (salbutamol 5 to 20 g/kg/d) and continuous sedation (benzodiazepine and neuromuscular blocking agent). MV was carried out according to the procedure of controlled hypoventilation in order to obtain the largest ventilation for a maximal airway pressure of 50 cm H 2 O. FI O2 was adapted to achieve Pa O2 60 mm Hg. Four patients without preexisting respiratory disease and requiring MV (V) were also studied to evaluate the effects of MV on the bronchial cellular events. These patients were admitted in ICU for drugsuicide attempt (benzodiazepine) and required short-lasting MV because of severe alveolar hypoventilation. No additional treatment was administered. Eleven patients with mild asthma were also investigated (A). Asthma was defined according to the criteria of the American Thoracic Society (12). All patients had either a reversible airway obstruction characterized by a 20% increase in forced expiratory volume in one second (FEV 1 ) after the inhalation of 200 g of albuterol or bronchial hyperreactivity to methacholine (PC 20 8 mg/ml). Exclusion criteria were inhaled or oral steroid use, history of an upper respiratory tract infection in the previous six weeks, severe exacerbation of asthma requiring hospitalization in the 6 wk preceding the study, and tobacco use within the past year or greater than 10 pack-yr total smoking history. The control group included eight healthy volunteers (C). They had no history of lung disease nor history of cigarette smoking. Their pulmonary function was within the normal range. The study was approved by the local ethic committee of the hospital. Characteristics of Asthma Sensitivity to aeroallergens was evaluated in all patients by skin prick tests. Atopy was defined as the process of at least two positive skin tests, i.e., wheal size superior to the one of histamine. None of the healthy control subjects had positive skin tests to aeroallergens. Serum total IgE were measured using the Phadebas Paper Radioimmunosorbent Test (Pharmacia Diagnostics, Fairfield, NJ). Asthma severity was assessed according to the scoring system of Aas (13): very mild forms received a score of 1 and incapacitating diseases requiring antiinflammatory medications received a score of 5. The grading is based on events that took place over a 1-yr period and considers both the symptoms (the number and duration of asthma episodes, total duration of symptoms, and presence or absence of symptom-free intervals between attacks) and the requirements for medications. Fiberoptic Bronchoscopy and Bronchial Lavage (BL) BL was performed in ventilated patients when it was expected to provide therapeutical benefit. Thus, BL were performed in case of characterized atelectasis (in group SA and V) and/or to remedy possible mucus impaction in patients with refractory SA despite optimal medical treatment. BL samples from patients with parenchymal opacities consistent with pneumonia on chest radiography were not included. Fiberoptic bronchoscopy was performed through an adaptor on the endotracheal tube designed to minimize air leak (Model ; Rüsch AG, Kernen, West Germany). The FI O2 was adjusted to 1.0 for 5 to 15 min before and throughout the procedure. The tip of the bronchoscope was wedged into different segmental bronchi to remove diffuse mucus impaction in patients without atelectasis or in the relevant segmental bronchus in patients with atelectasis. The lavage was performed by infusion of two or three 15-ml aliquots of sterile 0.9% saline solution at room temperature. Each aliquot was immediately gently aspirated and the various fractions were pooled. The patient was monitored closely while receiving an FI O2 of 1.0 for an additional 10 min. The FI O2 was then given back to the prebronchoscopy level as tolerated, using continuous pulse oximetry and arterial blood gases. In asthmatics and healthy patients, fiberoptic bronchoscopy was performed after local anesthesia with lidocane 2% applied to the upper respiratory tract. BL was performed in a segmental bronchus of the right middle lobe by slow infusion of two 15-ml aliquots of sterile 0.9% saline solution. Each aliquot was immediately gently aspirated and the different fractions were pooled. During bronchoscopy, oxygen was readily available and the patient had an intravenous infusion to provide venous access. Bronchial Lavage Processing After recovery, an aliquot of 5 ml was sent for quantitative and qualitative bacterial cultures (the diagnosis of low respiratory tract infection (LRTI) required 10 6 cfu/ml). BL was filtered through sterile surgical gauze to remove mucus. Lavage fluids (10 l) were used to determine the total cell counts using a standard hemacytometer. Aliquots of the BL (200 l) were cytocentrifuged and stained using eosin/ methylene blue (RAL, Paris, France) for differential cell counts. At least 500 cells on each slide were read by two investigators blinded to the clinical details of the patients. Averaged cell differentials of the two investigators are reported. Macrophages, neutrophils, lymphocytes, eosinophils, mast cells and epithelial cells were enumerated and results were given both in percentage and number of cells per millimeter of fluid recovered. The remainder of the lavage samples was centrifugated for 10 min at 400 g to separate the supernatant, which was removed and stored in aliquots at 70 C for later analysis. Evaluation of Mediator Concentrations Neutrophil elastase was evaluated by enzyme immunoassay as a complex with 1 -proteinase inhibitor (Merck KGaA, Darmstadt, Germany). In order to complex free elastase, purified 1 proteinase inhibitor (Sigma Co., St. Louis, MO) (2 mg/ml) was added to bronchial lavages before assay. Eosinophil Cationic Protein (ECP) was assayed in duplicate with specific and sensitive radioimmunoassays (Pharmacia, St. Quentin Yvelines, France). Histamine and IL-8 levels were measured by ELISA. Results were expressed as g/l, g/l, nm, and pg/ml for neutrophil elastase, ECP, histamine, and IL-8, respectively. The lower limits of detection for the assays evaluating neutrophil elastase, ECP, histamine, and IL-8 were 0.1 g/l, 1 g/l, 0.2 nm, and 10 pg/ml, respectively.

3 396 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL Statistical Analysis Group data are expressed as mean SEM and/or as median with interquartile range (IQR). Because most of the data were not normally distributed, statistical analysis was performed using the nonparametric Kruskall Wallis test for differences between the four groups. When this test indicated significant difference, each pairing was examined by means of Mann Whitney U test. The correlations between IL-8 and different cell types (neutrophils, eosinophils) were evaluated with Spearman s rank correlation coefficient test. A probability value of less than 0.05 was taken as statistically significant. RESULTS Clinical and Biological Data Clinical and biological characteristics of the patients are shown in Table 1. There were eight patients with status asthmaticus (SA) (mean age SEM, 33 4), four patients under MV without respiratory disease (V) ( ), 11 asthmatics (A) ( ) and eight healthy control subjects (C) ( ). There was no significant difference in age between the four groups. In SA group, there were four males and four females; two were exsmokers and one was current smoker; in group V, two were smokers whereas there was no former or current smoker in group A and in Group C. According to time elapsed from onset of symptoms of asthma attack to respiratory failure requiring MV, three patients with SA exhibited sudden-onset SA (interval time less than 3 h), and five progressive-onset SA (interval time superior to 3 h). Clinical data regarding probable events that triggered the asthma attack were available in five of eight patients with SA. In only two cases, a recent history of chest cold was identified. Nonspecific irritants triggered asthma attack in two cases and psychological conflicts in one case. Six patients with SA are still alive, whereas two died in ICU (Patients 4 and 5, respectively from pulmonary embolism at Day 30 and from nosocomial infection at Day 20). SA and A groups did not differ in terms of asthma severity and total IgE. Total IgE levels were significantly higher in SA and A than in C. Bronchial Lavage Sampling In SA group, the median number of bronchoscopy for BL per patient was two (range: one to four) (Table 2). BL were performed at different time intervals from onset of MV (Day 0 to Day 11) (median: Day 4). Indications for bronchoscopy were atelectasis associated or not with hypoxemia in seven patients and attempt to remove mucus impaction in nine patients. Neither death nor bronchoscopy-related complications (pneumothorax, pneumomediastinum, cardiac arrhythmias) were reported. In seven patients, BL resulted in marked improvement as assessed by decrease of maximal airway pressure and/or normalization of chest radiography. In five patients, no change was reported. In the remaining four, transient increase in maximal airway pressure with or without increase in Pa CO2 (less than TABLE 1 CLINICAL AND BIOLOGICAL DATA OF THE PATIENTS Subject No. Onset of SA* Age (yr) Sex Smoker (Yes/No) Probable Trigger Aas Score Atopy (SPT ) Total IgE (ku/l ) Patients with status 1 Sudden 41 F Y Chest cold 3 ND asthmaticus (n 8) 2 Sudden 23 F Y Cigarette smoke 2 ND 3 Sudden 46 M N NI/no history of chest cold Progressive 48 F N NI 1 ND 5 Progressive 24 M N NI/no history of chest cold 3 2,128 6 Progressive 38 F Y Chest cold 2 1,190 7 Progressive 19 M N Nonspecific (fog) 3 ND 8 Progressive 25 M N Psychological concerns 2 42 Patients under MV without 1 32 M Y ND respiratory disease (n 4) 2 43 M Y ND 3 38 M N ND 4 52 M N ND Patients with stable 1 65 M N asthma (n 11) 2 40 F N M N 2 1, M N M N M N M N M N F N F N M N 2 10 Controls (n 8) 1 19 M N M N M N M N F N F N M N F N 12 Definition of abbreviations: SA status asthmaticus; MV mechanical ventilation; NI not identified; SPT skin prick tests; ND not done. * According to time from onset of asthma symptoms to onset of mechanical ventilation: less than 3 h sudden, superior to 3 h progressive onset of SA. Positive skin prick tests to two or more inhalant antigens.

4 Lamblin, Gosset, Tillie-Leblond, et al.: Bronchial Neutrophilia in Status Asthmaticus 397 TABLE 2 BRONCHIAL LAVAGE CHARACTERISTICS IN PATIENTS WITH STATUS ASTHMATICUS Subject No. Number of BL Day* Chest Radiograph Bacteriological Examination 1 1 D1 Right lower lobe atelectasis P. mirabilis 10 7 cfu/ml 2 4 D1 Normal Sterile D4 Lamellar bilateral atelectasis Sterile D5 Right lower lobe atelectasis Sterile D9 Right lower lobe atelectasis P. aeruginosa 10 8 cfu/ml 3 1 D0 Normal Sterile 4 2 D0 Normal Sterile D1 Left lower lobe atelectasis Sterile 5 2 D5 Right lower lobe atelectasis Sterile D6 Normal Sterile 6 3 D0 Normal Sterile D1 Normal Sterile D4 Normal S. pneumoniae 10 6 cfu/ml 7 2 D4 Normal Sterile D11 Left lower lobe atelectasis Sterile 8 1 D6 Normal Sterile Definition of abbreviations: BL bronchial lavage; cfu colony forming unit. * Time elapsed from onset of mechanical ventilation to BL sampling. 10 mm Hg) was reported. LRTI was suspected to be the trigger of the asthma attack in two patients (Patients 1 and 6, Table 2). In Patient 2, BL grew P. aeruginosa 10 8 cfu/ml on Day 9 (nosocomial LRTI). The remaining 13 BL samples were sterile. In group V, BL were performed within the first 48 h of MV (between the 12th to the 48th h of MV). All BL were sterile. In groups V, A, and C, no complication related to bronchoscopy occurred. BL Cellularity Total cell number was significantly higher in SA than in V, A, and in C (Figure 1). Detailed data regarding differential cellular counts in SA are displayed in Table 3. SA exhibited marked increase in number and percentage of neutrophils as compared with V, C, and A (Figure 1 and Table 4). Eosinophils represented more than 5% of cells in 5 BL from patients with SA. Although there was a trend for percentage of eosinophils to be higher in SA than in A, this difference did not reach significance. Number of eosinophils was significantly increased in BL from SA as compared with A. Percentage and number of eosinophils were significantly higher in SA than in V and C. Percentage of eosinophils was higher in group A than in V and C. Number and/or percentage of mast cells did not differ between the four groups, although they tended to be higher in BL from SA (data not shown). SA demonstrated lower percentage of macrophages and lymphocytes than V, A, and C whereas total number of lymphocytes were not significantly different. Percentages of epithelial cells recovered in BL did not differ between the four groups, whereas total number of epithelial cells was increased in SA in comparison with C (p 0.05). Figure 1. Total cell counts and differential counts in bronchial lavage in the four groups of patients. Data presented as box plots. Horizontal lines represent the median, squares the 25th and 75th percentiles, and bars the 10th and 90th percentiles. C controls (n 8); A mild asthmatics (n 11); V patients under mechanical ventilation without respiratory disease (n 4); SA status asthmaticus (n 16). *p 0.05; p 0.01; p 0.005; p ; p Levels of Neutrophil Elastase, ECP, Histamine and Interleukin-8 SA group showed a marked increase in neutrophil elastase levels as compared with V, A and C (Figure 2). ECP levels were significantly higher in SA than in V, A, and C. Histamine was higher in SA than in V and C. Histamine tended to be higher in SA than in A but this difference did not reach statistical significance (p 0.09). IL-8 was markedly increased in SA as compared with V, A, and C. The neutrophil count in all patients significantly positively correlated with IL-8 levels

5 398 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL TABLE 3 TOTAL CELL COUNTS AND CELL PERCENTAGES IN BRONCHIAL LAVAGE FROM PATIENTS WITH STATUS ASTHMATICUS Subject No. Day* Total Cell Count (10 6 /ml ) Neutrophils Macrophages Lymphocytes Eosinophils Mast Cells Epithelial Cells 1 D D D D D D D D D D D D D D D D Mean SEM Median (IQR) 1.05 (1.01) 88.3 (19.1) 2.4 (2.8) 0.3 (0.4) 1.3 (5.0) 0.0 (0.2) 2.0 (14.1) Definition of abbreviation: BL bronchial lavage. * Time elapsed from onset of mechanical ventilation to BL sampling. (r s 0.57; p ). Number of eosinophils positively correlated with IL-8 levels (r s 0.37; p 0.03). Concommitant Low Respiratory Tract Infection Data concerning total cell number and differential cellular counts in BL with positive bacteriological cultures (n 3) and sterile BL (n 13) are disclosed in Table 5. Total cell count was higher in sterile BL than in BL with positive bacteriological cultures, whereas differential cellular count was similar in both groups. Similarly, neutrophil elastase, ECP, histamine, and IL-8 levels did not differ between BL with positive bacteriological cultures and sterile BL. MV (p 0.06). In the latter, histamine was statistically increased (p 0.05). Bronchial neutrophilia and neutrophil elastase levels were similar whatever the delay from onset of MV to BL sampling. Total cell count, macrophages, lympho- Time Elapsed from Onset of Mechanical Ventilation to BL Sampling BL performed within the first 48 h of mechanical ventilation (before Day 2) exhibited a higher number and percentage of eosinophils (p 0.05), and mast cells (p 0.05), as compared with BL performed after 48 h of MV (Table 6). ECP levels tended to be higher in BL performed within the first 48 h of TABLE 4 CELL NUMBERS IN BRONCHIAL LAVAGE IN THE FOUR GROUPS OF PATIENTS SA V A C Total cell count 1.05 (1.01)* 0.31 (0.48) 0.13 (0.45) 0.12 (0.28) Neutrophils 0.87 (0.98)* 0.17 (0.32) 0.01 (0.02) 0.00 (0.05) Macrophages 0.03 (0.06)* 0.11 (0.14) 0.09 (0.30) 0.08 (0.08) Lymphocytes 0.00 (0.00) 0.00 (0.01) 0.01 (0.04) 0.00 (0.01) Eosinophils 0.00 (0.07) 0.00 (0.00) 0.00 (0.00) 0.00 (0.00) Epithelial cells 0.05 (0.06) 0.04 (0.04) 0.01 (0.02) 0.01 (0.02) Definition of abbreviations: C controls (n 8); A mild asthmatics (n 11); V patients under mechanical ventilation without respiratory disease (n 4); SA status asthmaticus (n 16). Results are expressed as numbers of cells per 10 6 /ml. Data are shown as medians with interquartile range in parentheses. * p 0.05 and p 0.01, statistically different from V. p 0.05, statistically different from A. p 0.05, statistically different from C. p 0.01 and p 0.001, statistically different from A and C. Figure 2. Neutrophil elastase, eosinophil cationic protein (ECP), histamine and interleukin-8 (IL-8) levels in bronchial lavage in the four groups of patients. Data presented as box plots. Horizontal lines represent the median, squares the 25th and 75th percentiles, and bars the 10th and 90th percentiles. C controls (n 8); A mild asthmatics (n 11); V patients under mechanical ventilation without respiratory disease (n 4); SA status asthmaticus (n 16). *p 0.05; p 0.01; p 0.005; p 0.001; p

6 Lamblin, Gosset, Tillie-Leblond, et al.: Bronchial Neutrophilia in Status Asthmaticus 399 TABLE 5 BRONCHIAL LAVAGE CHARACTERISTICS ACCORDING TO BACTERIOLOGICAL CULTURES IN PATIENTS WITH STATUS ASTHMATICUS Bacteriological Cultures* Positive (n 3) Sterile (n 13) Total cell count 10 6 /ml 0.96 (0.65) 1.07 (1.48) Neutrophils % 90.0 (11.8) 86.7 (27.8) 10 6 /ml 0.86 (0.69) 0.88 (1.42) Macrophages % 2.0 (4.1) 2.5 (2.0) 10 6 /ml 0.03 (0.04) 0.03 (0.06) Lymphocytes % 0.1 (0.1) 0.4 (0.5) 10 6 /ml 0.00 (0.00) 0.00 (0.01) Eosinophils % 1.0 (4.2) 1.7 (4.9) 10 6 /ml 0.01 (0.02) 0.01 (0.10) Mast cells % 1.0 (4.2) 0.0 (0.01) 10 6 /ml 0.00 (0.00) 0.00 (0.00) Epithelial cells % 1.0 (8.2) 2.0 (19.8) 10 6 /ml 0.01 (0.04) 0.07 (0.06) Neutrophil elastase g/l (202.5) (405.0) ECP g/l 98.0 (44.2) (142.2) Histamine nm/l 15.5 (34.6) 14.5 (19.0) IL-8 pg/ml (289.5) (545.0) Definition of abbreviations: ECP eosinophil cationic protein; IL-8 interleukin-8; IQR interquartile range. Results are expressed as median (IQR). * Bacteriological cultures were taken as positive when bacteriological count was 10 6 cfu/ml. cytes, and epithelial cells were similar in BL performed within the first 48 h and in BL performed later. ECP levels in BL performed within the first 48 h of MV were higher than A (p ) and remained higher than A in BL performed later (p ), whereas percentage of eosinophils in BL performed after 48 h of MV did not differ from A (data not shown). Onset of Symptoms of Asthma Attack to Respiratory Failure We categorized patients with SA according to the time interval from onset of asthma attack to respiratory failure leading TABLE 6 BRONCHIAL LAVAGE CHARACTERISTICS IN PATIENTS WITH STATUS ASTHMATICUS ACCORDING TO TIME ELAPSED FROM ONSET OF MV TO BL SAMPLING Interval Time* 48 h (n 7) 48 h (n 9) p Value Total cell count 10 6 /ml 1.18 (3.56) 0.55 (1.00) 0.18 Neutrophils % 91.0 (7.0) 86.0 (29.7) /ml 1.00 (3.74) 0.44 (1.08) 0.26 Macrophages % 2.5 (4.6) 2.3 (2.0) /ml 0.04 (0.07) 0.02 (0.03) 0.12 Lymphocytes % 0.4 (0.9) 0.3 (0.3) /ml 0.00 (0.01) 0.00 (0.00) 0.28 Eosinophils % 5.3 (7.1) 1.0 (1.1) /ml 0.09 (0.31) 0.00 (0.00) 0.03 Mast cells % 0.3 (0.8) 0.0 (0.0) /ml 0.00 (0.01) 0.00 (0.00) 0.05 Neutrophil elastase g/l 1,050.0 (405.0) (550.0) 0.79 ECP g/l (111.7) 98.0 (138.0) 0.06 Histamine nm/l 45.5 (54.6) 12.0 (11.3) 0.05 IL-8 pg/ml (858.0) (326.7) Definition of abbreviations: MV mechanical ventilation; ECP eosinophil cationic protein; IL-8 interleukin-8; IQR interquartile range. Results are expressed as median (IQR). * Time elapsed from onset of MV to BL sampling. Probability value of Mann Whitney U test performed between BL sampled within the first 48 h and BL performed after 48 h of MV (p 0.05 was taken as statistically significant). to MV. We compared total cell number and differential cell count in the first BL performed in patients with sudden-onset asthma attack (n 3) and in patients with progressive-onset asthma attack (n 5). Small number of BL in each group did not allow statistical analysis. Total cell count, as well as percentage and number of neutrophils, tended to be higher in patients with sudden-onset asthma attack, whereas eosinophils, macrophages and lymphocytes were similar (Table 7). Neutrophil elastase, ECP, and IL-8 levels were also similar in both groups, whereas histamine tended to be higher in BL from patients with sudden-onset asthma attack. DISCUSSION The present study was designed to evaluate cellular effectors that were implicated during the course of SA. Our results clearly demonstrated a prominent bronchial neutrophilia in BL samples from patients in SA, at early and late stages of SA. Bronchial neutrophilia was associated with increased number of eosinophils and mast cells at the initial stage of SA. These pathological changes were not related to bacterial LRTI. The prominent neutrophilia may be linked, at least in part, to an increased level of IL-8 measured in BL samples. Bronchial neutrophilia in the course of SA is a novel and questionable finding. To our knowledge, data regarding cell types implicated in SA were merely available in postmortem studies (3 9). We had the opportunity to analyze cellular components of BL obtained during bronchoscopy in patients undergoing MV for SA. BL and/or BAL are not a routine procedure in SA. Their indications are not precisely defined in this at-risk situation. Efficacy and safety of BL performed during SA are not validated by prospective and retrospective studies for now. Nonetheless, according to ATS recent guidelines concerning the management of SA (11), BL may be useful to remove bronchial obstruction due to the constant impaction of mucous plugs. Lang and colleagues demonstrated that BAL may improve bronchial obstruction, as assessed by FEV 1 and TABLE 7 CHARACTERISTICS OF THE FIRST BRONCHIAL LAVAGE IN PATIENTS WITH STATUS ASTHMATICUS ACCORDING TO TIME FROM ONSET OF SYMPTOMS TO ONSET OF MV Onset of SA* Progressive (n 5) Sudden (n 3) Total cell count 10 6 /ml 0.34 (0.96) 1.38 (3.48) Neutrophils % 57.0 (43.8) 90.0 (7.2) 10 6 /ml 0.25 (0.44) 1.17 (3.32) Macrophages % 3.0 (7.1) 2.5 (4.9) 10 6 /ml 0.02 (0.04) 0.04 (0.02) Lymphocytes % 0.5 (1.9) 0.2 (0.1) 10 6 /ml 0.00 (0.00) 0.00 (0.01) Eosinophils % 2.0 (9.8) 1.7 (7.2) 10 6 /ml 0.01 (0.10) 0.09 (1.00) Mast cells % 0.0 (0.1) 0.8 (0.4) 10 6 /ml 0.00 (0.00) 0.01 (0.02) Neutrophil elastase g/l (270.0) 1,010.0 (345.0) ECP g/l (47.5) (104.2) Histamine nm/l 14.5 (32.0) 45.5 (22.1) IL-8 pg/ml (1192.2) (13.5) Definition of abbreviations: MV mechanical ventilation; ECP eosinophil cationic protein; IL-8 interleukin-8; IQR interquartile range. Results are expressed as median (IQR). * Time elapsed from onset of symptoms of asthma attack to respiratory failure leading to MV (progressive interval time 3 h; sudden interval time 3 h). Probability value of Mann Whitney U test performed between BL from patients with progressive-onset SA and BL from patients with sudden-onset SA (p 0.05 was taken as statistically significant).

7 400 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL FEF measurements, in nonintubated patients with stable but refractory acute asthma, without major complications (14). We performed bronchoscopy and BL sampling in case of refractory SA despite optimal medical treatment, in order to remedy possible mucus impaction, or in case of atelectasis. In order to minimize complications, samples were obtained in segmental bronchi, during rapid bronchoscopy with small volumes of fluid and were likely representative of proximal airways, rather than alveolar spaces. We did not notice any severe complications, such as barotraumatism and/or cardiac arrhythmias, related to bronchoscopy and minor complications (transient increase of airway pressure and/or transient increase of Pa CO2 ) were observed in four of 16 cases. It is of interest to point out that clinical improvement was obtained in seven cases. The first striking point of our study consists with a marked predominance of neutrophils over eosinophils in BL from patients with SA. Until now, eosinophils were thought to be the predominant cells of SA as they were described in several autopsy studies of fatal asthma cases (15). In addition to shedding of the bronchial epithelium, thickening of the basement membrane, hypertrophy of bronchial smooth muscle, airways infiltration by activated eosinophils was usually demonstrated in several studies of fatal asthma cases (8, 9). Eosinophils were also demonstrated filling the mucus in patients dying in SA (3, 4). Our finding of a moderate eosinophilia in SA may appear as inconsistent with previous studies. However, it has to be noticed that the present study and the previous ones are not strictly comparable. First, autopsic studies may miss dynamic changes that occur during successive stages of SA. Indeed, we reported varying eosinophil and mast cell numbers according to time elapsed from onset of MV. Second, autopsic studies focused on cellular infiltrate into the airways submucosa and on lung parenchyma, corresponding with tissular areas, whereas we reported inflammatory changes within the lumen of proximal airways. The discrepancy between autopsic studies and our study may indicate a preferential localization of eosinophils in the bronchial mucosa and in lung parenchyma rather than in airway lumen of patients in SA, that we only evaluated in this study. We believe that eosinophil count was underestimated in the present study and that eosinophils are central to SA pathogenesis. This is supported by several data. First, dramatic increased ECP levels were detected in the absence of a pronounced increase of eosinophil count in BL, at early stage of SA. Moreover, ECP levels remained highly elevated at later stage of SA, whereas eosinophil number was apparently within normal values. In fact, activated and degranulated eosinophils may be missed when a counting method dependent on staining is used, minimizing actual eosinophil count (16). Unfortunately, we were unable to perform immunohistochemistry on BL cells from SA due to a nonspecific binding of monoclonal antibodies on these slides probably related to the presence of mucus. Secondly, our patients with SA were all receiving parenteral corticosteroids before and during MV. This therapy is prone to induce a decrease of bronchial eosinophil count as it was previously reported that blood, alveolar and sputum eosinophils were sensitive to corticosteroids (17, 18). Thirdly, samples were carried out in segmental bronchi, that is to say in proximal airways, with small volumes of fluid; the two latter factors may also affect eosinophil count by increasing neutrophil count (19, 20). In addition, significant increase of mast cells and histamine levels, in association with increase of eosinophils and ECP, was disclosed in BL performed within the first 48 h of MV. This data is consistent with the classic role of mast cells in the early-phase asthmatic reaction. Indeed, mast cells and their mediators have been previously found in increased numbers in BAL of both atopic and nonatopic asthmatic subjects. Moreover, mast cell percentage was reported to decrease during allergen-induced late-phase reactions (2). Our findings support the hypothesis of an active participation of eosinophils and mast cells at early stage of SA. Thus, increase of eosinophils and mast cells may constitute the early step in the cellular events that occur in the course of SA. Bronchial prominent neutrophilia was noticeable as it occurred to be constant and homogenous in all BL from patients with SA. This data is in keeping with two recent autopsic studies which reported increased neutrophils in fatal asthma cases, especially in cases of sudden-onset of fatal asthma. Carroll demonstrated that, in cases of fatal attacks of short duration (time interval 2 h), the neutrophil count was increased and the numbers of eosinophils were reduced as compared to cases with fatal attacks of long duration (time interval 5 h) (6). In Sur and coworkers study, immunofluorescence assays of bronchial submucosa in sudden-onset fatal asthma (time interval less than 1 h) revealed significantly more neutrophils and fewer eosinophils that did slow-onset fatal cases (time interval 2.5 h), without evidence of upper respiratory tract infection (7). At variance with our findings, Sur reported that number of eosinophils tended to increase with the increase of time between the onset of asthma attack and death. Although small number of patients did not allow statistical comparison, we also reported that percentage and total number of neutrophils tended to be higher in patients with sudden-onset SA in comparison with progressive-onset SA while no difference in eosinophil count was observed between the two groups of patients. This point will need further investigation with higher number of patients. Additional factors may also affect cellular events that occur within bronchial lumen in patients undergoing MV for SA. Thus, we raised they hypothesis that bronchial neutrophilia might not be specific of SA and was related either to concomitant bacterial respiratory infection or to the institution of therapy for SA, such as MV and corticosteroids. Participation of a concomitant bacterial infection was considered. Indeed, Fahy (10) demonstrated that neutrophils were the predominant inflammatory cells in sputum from 18 patients with asthma in acute exacerbation. Respiratory tract infection was thought to be the trigger of asthma exacerbation in eight cases, although no objective measurements were performed to assess bacteriological infection. Turner (21) displayed that almost half of 34 subjects considered to have mild uncontrolled asthma had no increase of sputum eosinophilia and rather exhibited an increase in sputum neutrophilia, without evidence of respiratory tract infection. In our study, respiratory tract infection was systematically investigated and was thought to be responsible for the onset of SA in two cases. Moreover, cellular components of BL with positive bacteriological cultures did not differ from sterile BL. Thus, respiratory tract infection did not appear to be the main precipitating factor of SA and did not account for the marked bronchial neutrophilia. We also investigated the contribution of MV to bronchial cellular changes. It appears that MV may affect bronchial neutrophilia as we noticed a significant increase of neutrophil count in patients without preexisting respiratory disease undergoing MV as compared with patients who were not submitted to MV. However, bronchial neutrophilia remained significantly lower than in patients undergoing MV for SA, suggesting that bronchial neutrophilia was quite specific of SA. This finding of a moderate neutrophilia related to MV is supported by a previous study which demonstrated that nonasthmatic patients un-

8 Lamblin, Gosset, Tillie-Leblond, et al.: Bronchial Neutrophilia in Status Asthmaticus 401 dergoing MV exhibited a moderate neutrophilia (22). It is true that we studied the effects of MV in a small number of patients and only within the first 48 h of MV. It would be of interest to study long-term effects of MV. However, most of patients requiring long-term MV actually have either a preexisting respiratory disease or have developed systemic or respiratory infection, accounting for the difficulty to assess specific long-term effects of MV on airways cellular components. The influence of systemic steroids given during the course of SA has also to be considered. Systemic steroids are known to be potent anti-inflammatory agents that may modify inflammatory cell profiles within the airways of asthmatics. It is now certain that corticosteroids may induce a decrease of eosinophils either in sputum or in bronchial biopsies from asthmatics (17, 18). On the other hand, steroids are prone to activate neutrophil progenitors and to increase circulating neutrophils but their effects on bronchoalveolar neutrophils are less clear. Djukanovic recently reported no change in neutrophil count in bronchoalveolar lavage and in bronchial biopsies in asthmatic patients treated with 20 mg of prednisolone during two weeks (18). Similar findings were reported by Dworsky (23). Conversely, neutrophil percentage was reported to be moderately increased after 1 yr of continuous treatment with steroids in patients developing corticodependent asthma, whereas eosinophils decreased (24). Nonetheless, some patients had elevated neutrophil counts before steroid treatment was started, suggesting that neutrophilia may be related to disease severity. Tanizaki also demonstrated that corticodependent asthmatics exhibited modest increased neutrophils (mean, 12.5%) in broncho-alveolar lavage as compared with mild asthmatics (25). Lastly, in another inflammatory respiratory disease such as idiopathic pulmonary fibrosis, long-term steroid therapy was reported to be associated with a slight increase of neutrophil percentage in smoking patients (26). Taken together, these results suggest the hypothesis that dramatic increase of neutrophils in SA was related to the disease severity instead of steroid therapy. Lastly, it is possible to raise the hypothesis that bronchial neutrophilia was due to the inhalation of ubiquitinous endotoxins and/or Alternaria. Both factors have been reported to induce recruitment of neutrophils (27, 28), but implication of these factors as triggers of asthma attacks appears delicate to assess, especially in clinical situations. The significance of this marked neutrophilia remain unclear, as the role of neutrophils in asthma is still poorly understood (29). Several studies in animals and/or in man argue for the neutrophil to be especially involved in asthma exacerbations and experimentally induced airway hyperresponsiveness. In primates spontaneously allergic to Ascaris suum, allergen challenge induced large influx of neutrophils and activated eosinophils in association with the occurrence of late phase airway obstructive response. Blocking the neutrophil influx by anti E-selectine monoclonal antibodies inhibited the late phase airway obstruction (30). In guinea-pig sensitized to A. suum or ovalbumine, similar findings concerning neutrophils and eosinophils recruitment after allergen challenge were reported (31). In man, bronchoalveolar neutrophilia was reported before, during and after late asthmatic reactions induced either by toluene diisocyanate or by allergens, following or concomitant to bronchoalveolar eosinophilia (29). At variance with these studies, we did not investigate the distal airways as we reported cellular changes present in proximal airways. However, these observations and our study suggest that there is at least an association between the migration of neutrophils in the airway spaces and asthma attacks and/or the occurrence of SA. Of relevance to possible mechanisms of neutrophilic infiltration of proximal airways in SA is our finding of elevated IL-8 levels in SA. Neutrophil count and IL-8 levels were positively correlated. Recruitment of neutrophils in SA was previously reported by Corrigan to be secondary to release of a neutrophil chemotactic factor, even if it was described as distinct from IL-8 (32). Concerning IL-8, it is known to be a potent chemoattractant and activating cytokine for neutrophils, and to a lesser extent for eosinophils. Endothelial cells, fibroblasts, epithelial cells, alveolar macrophages and neutrophils are able to release IL-8 in response to specific stimuli (TNF, IL-1, and endotoxin) (33). In case of SA, neutrophil recruitment may result from an inflammatory cascade that begins with airway injury (inhalation of aeroirritants, aeroallergen, endotoxin, infection), which is followed by IL-8 secretion by endothelial cells, epithelial cells and alveolar macrophages. This proceeds to chemoattraction and activation of neutrophils, which may release several products that acutely damage airways structure. Indeed, neutrophils are well equipped to alter airways function by the release of biologically patent preformed granule associated mediators (neutrophil elastase, metalloproteinases), as well as products of de novo synthesis (oxidative products, eicosanoids) (34). Interestingly, neutrophil elastase is a potent secretagogue of respiratory glycoconjugates by submucosal glands and goblet cells which form, in association with proteins and cells, solid mucus plugs that may be responsible for refractory airways obstruction observed in SA (35). In conclusion, our study provides new insights allowing to suspect the involvement of neutrophils to SA pathogenesis. We displayed a cellular infiltration of the bronchial compartment with neutrophils and eosinophils in SA, which differs from that seen in mild asthma. Increased levels of IL-8 might partly explain neutrophil migration to bronchial lumen. However, precise mechanisms of neutrophil recruitment to the airways in SA remain to be elucidated. Assessment of other chemokines production and evaluation of adhesion molecules expression on endothelial cells and other inflammatory cells during SA should provide a better understanding of the mechanisms implicated during the course of SA. References 1. Bousquet, J., P. Chanez, J. Y. Lacoste, B. Barnéon, N. Ghavanian, I. Enander, P. Venge, S. Ahlstedt, J. Simony-Lafontaine, P. Godard, and F.-B. Michel Eosinophilic inflammation in asthma. N. Engl. J. Med. 323: Smith, D. L., and R. D. Deshazo Bronchoalveolar lavage in asthma: an update and perspective. Am. Rev. Respir. Dis. 148: Cardell, B. S., and R. S. B. Pearson Death in asthmatics. Thorax 14: Houston, J. C., S. De Navasquez, and J. R. Trounce A clinical and pathological study of fatal cases of status asthmaticus. Thorax 8: Carroll, N., J. Elliot, A. Morton, and A. James The structure of large and small airways in non fatal and fatal asthma. Am. Rev. Respir. Dis. 147: Carroll, N., S. Carello, C. Cooke, and A. James Airway structure and inflammatory cells in fatal attacks of asthma. Eur. Respir. J. 9: Sur, S., T. B. Crotty, G. M. Kephart, B. A. Hyma, T. V. Colby, C. E. Reed, L. W. Hunt, and G. J. Gleich Sudden-onset fatal asthma: distinct entity with few eosinophils and relatively more neutrophils in the airway submucosa? Am. Rev. Respir. Dis. 148: Synek, M., R. Beasley, A. J. Frew, D. Goulding, L. Holloway, F. C. Lampe, W. R. Roche, and S. T. Holgate Cellular infiltration of the airways in asthma of varying severity. Am. J. Respir. Crit. Care Med. 154: Azzawi, M., P. W. Johnston, S. Majumdar, A. B. Kay, and P. K. Jeffery T lymphocytes and activated eosinophils in airway mucosa in fatal asthma and cystic fibrosis. Am. Rev. Respir. Dis. 145: Fahy, J. V., K. W. Kim, J. Liu, and H. A. Boushey Prominent neutrophilic inflammation in sputum from subjects with asthma exacerba-

9 402 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL tion. J. Allergy Clin. Immunol. 95: Corbridge, T. C., and J. B. Hall The assessment and management of adults with status asthmaticus. Am. J. Respir. Crit. Care Med. 151: American Thoracic Society Standards for the diagnosis and care of patients with chronic obstructive pulmonary disease (COPD) and asthma. Am. Rev. Respir. Dis. 136: Aas, K Heterogeneity of bronchial asthma: subpopulations or different stages of the disease. Allergy 36: Lang, L. M., R. A. Simon, D. A. Mathison, R. M. Timms, and D. D. Stevenson Biopsy and possible efficacy of fiberoptic bronchoscopy with lavage in the management of refractory asthma with mucous impaction. Ann. Allergy 67: Gleich, G. J., S. Motojima, E. Frigas, G. M. Kephart, T. Fjuisawa, and L. P. Kravis The eosinophilic leucocyte and the pathology of fatal bronchial asthma: evidence for pathologic heterogeneity. J. Allergy Clin. Immunol. 80: Leiferman, K. M., S. J. Ackerman, H. A. Sampson, H. S. Haugen, P. Y. Venencie, and G. J. Gleich Dermal deposition of eosinophilgranule major basic protein in atopic dermatitis. N. Engl. J. Med. 313: Claman, D. M., H. A. Boushey, J. Liu, H. Wong, and J. V. Fahy Analysis of induced sputum to examine the effects of prednisone on airway inflammation in asthmatic patients. J. Allergy Clin. Immunol. 94: Djukanovic, R., S. Homeyard, C. Gratziou, J. Madden, A. Walls, S. Montefort, D. Peroni, R. Polosa, S. Holgate, and P. Howarth The effect of treatment with oral corticosteroids on asthma symptoms and airway inflammation. Am. J. Respir. Crit. Care Med. 155: Tonnel, A. B., C. Voisin, J. J. Lafitte, P. Ramon, and C. Aerts Variations des populations cellularies recueillies par lavage bronchoalvéolaire en fonction de la topographie des lésions et de l étage exploré. In G. Biserte, J. Chrétien, and C. Voisin, editors. Le Lavage Broncho-alvéolaire. INSERM ed, Paris. 34: Lam, S., J. C. Leriche, K. Kijek, and D. Phillips Effect of bronchial lavage volume on cellular and protein recovery. Chest 88: Turner, M. O., P. Hussack, M. R. Sears, J. Dolovich, and F. E. Hargreave Exacerbations of asthma without sputum eosinophilia. Thorax 50: Delclaux, C., M. P. D ortho, C. Delacourt, F. Lebargy, C. Brun-Buisson, L. Brochard, F. Lemaire, C. Lafuma, and A. Harf Gelatinase in epithelial lining fluid of patients with adult respiratory distress syndrome. Am. J. Physiol. (Lung Cell. Mol. Physiol.) 272: Dworsky, R., G. A. Fitzgerald, J. A. Oates, and J. R. Sheller Effect of oral prednisone on airway inflammatory mediators in atopic asthma. Am. J. Respir. Crit. Care Med. 149: Chanez, P., A. M. Vignola, L. Paradis, A. Des Roches, P. Vic, P. Godard, and J. Bousquet Changes in airways inflammation in patients developing corticodependent asthma (abstract). Am. J. Respir. Crit. Care Med. 153:A Tanizaki, Y., H. Kitani, M. Okazaki, T. Mifune, F. Mitsunobu, and I. Kimura Changes in the proportion of bronchoalveolar lymphocytes, neutrophils and basophilic cells and the release of histamine and leukotrienes from bronchoalveolar cells in patients with steroiddependent intractable asthma. Int. Arch. Allergy Immunol. 101: Watters, L. C., T. E. King, R. M. Cherniack, J. A. Waldron, R. E. Sanford, M. L. Willcox, K. L. Christopher, and M. I. Schwarz Bronchoalveolar lavage fluid neutrophils increase after corticosteroid therapy in smokers with idiopathic pulmonary fibrosis. Am. Rev. Respir. Dis. 133: Hunt, L. W., E. S. Mansfiels, S. Sur, and G. J. Gleich Late neutrophilic response to bronchial allergen challenge: a response to endotoxin? J. Allergy Clin. Immunol. 89: O Hollaren, M. T., J. W. Yuginger, K. P. Offord, M. J. Sommers, E. J. O Connel, D. J. Ballard, and M. I. Sachs Exposure to an aeroallergen as a possible precipitating factor in respiratory arrest in young patients with asthma. N. Engl. J. Med. 324: Boschetto, P., C. E. Mapp, G. Picotti, and L. M. Fabbri Neutrophils and asthma. Eur. Respir. J. 6(Suppl.):S456 S Gundel, R. H., C. D. Wegner, C. A. Torcenelli, C. C. Clarke, N. Haynes, R. Rothlein, C. W. Smith, and L. G. Letts Endothelial-leucocyte adherence molecule-1 mediates antigen-induced acute airway inflammation and late phase obstructive response. J. Clin. Invest. 88: Hutson, P. A., M. K. Church, T. P. Clay, P. Miller, and S. T. Holgate Early and late-phase bronchoconstriction after allergen challenge of non anesthetized guinea pigs: I. The association of disordered airway physiology to leukocyte infiltration. Am. Rev. Respir. Dis. 137: Corrigan, C. J., P. Collard, L. Nagy, and A. B. Kay Status asthmaticus spontaneously elaborate a neutrophil chemotactic activity distinct from interleukin-8. Am. Rev. Respir. Dis. 143: Strieter, R. M., N. W. Lukacs, T. J. Standiford, and S. L. Kunkel Cytokines and lung inflammation: mechanisms of neutrophil recruitment to the lung. Thorax 48: Weiss, S. J Mechanisms of disease: tissue destruction by neutrophils. N. Engl. J. Med. 320: Sheehan, J. K., P. S. Richardson, D. C. K. Fung, M. Howard, and D. J. Thornton Analysis of respiratory mucus in asthma: a detailed study from a patient who died in status asthmaticus. Am. J. Respir. Cell Mol. Biol. 13: