Sputum in Severe Exacerbations of Asthma Kinetics of Inflammatory Indices after Prednisone Treatment

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1 Sputum in Severe Exacerbations of Asthma Kinetics of Inflammatory Indices after Prednisone Treatment MARCIA M. M. PIZZICHINI, EMILIO PIZZICHINI, LYNDA CLELLAND, ANN EFTHIMIADIS, JAMES MAHONY, JERRY DOLOVICH, and FREDERICK E. HARGREAVE Asthma Research Group, Departments of Medicine and Paediatrics, St. Joseph's Hospital and McMaster University, Hamilton, Ontario, Canada We have investigated the time -course of symptoms, forced expiratory volume in one second (FEV 1 ), and the airway inflammatory changes in sputum selected from saliva and blood of 10 patients with severe exacerbation of asthma betwen presentation and after 1, 2, 3, 7, and 21 days of treatment. The sputum was induced by a modified standard protocol, and we examined its safety. The severe exacerbation of asthma was defined by the presence of nocturnal symptoms disturbing sleep and/or the need for inhaled short acting (32-agonist >_ 8 puffs/d and an FEV, after bronchodilator < 60% of predicted. The treatment consisted of additional prednisone 30 mg daily for 5 d followed by reduction to zero by day 10. Abnormal findings [median (interquartile range)] in spontaneous and induced sputum included low viability of cells [52.0 (34.0)%]; eosinophilia [20.0 (16.4)%]; many free eosinophil granules; and increased levels of fluid-phase ECP [1960 (9204) µg/l], fibrinogen [6045 (10720) p.g/l], and IL-5 [160 (212) pg/ml]. Peripheral blood eosinophils [10.4 (7.6) %] and ECP levels [34.0 (35.0) µg/l] were increased. After treatment, symptoms, FEV I, blood eosinophilia, and serum ECP improved in the first 24 h. Sputum eosinophils and ECP did not improve until 48 h and fibrinogen not until 7 d. The improvement in sputum eosinophils and ECP levels was correlated with improvement of FEVI and in fluid-phase IL-5. Thirty sputum inductions were performed safely in the majority with inhaled isotonic or 3% saline (23.3% or 63.3%, respectively) over a short duration (mean 8.4 min). The patients who had a fall in FEVI of > 10% (10 occasions) after induction differed from those with a fall of < 10% only in the amount of inhaled (3 2-agonist used by the patients in the preceding 24 h [8.0 (5.0) versus 4.0 (3.0) puffs/d, p = 0.01]. The results suggest that spontaneous or induced sputum can be used safely to follow the kinetics of effects of antiinflammatory treatment in a severe exacerbation of asthma. The clinical and blood indices improve before those in sputum, raising the possibility that examination of sputum is a better guide in these patients to follow the effects of treatment. Pizzichini MMM, Pizzichini E, Clelland L, Efthimiadis A, Mahony J, Dolovich J, Hargreave FE. Sputum In severe exacerbations of asthma: kinetics of inflammatory indices after prednisone treatment. AM J RESPIR CRIT CARE MED 1997;155: Airway inflammation is considered to be the cause of asthma and an important determinant of exacerbations, current severity, and prognosis. The inflammation has been studied directly by bronchoscopy with bronchial biopsies, bronchoalveolar layage (BAL) (1, 2), sputum examination (3-5), and, indirectly, by measurements in peripheral blood (6,7). Until recently, sputum examination in asthma was considered unsuitable because the results were unreliable (8). Studies involving bronchoscopy are invasive and therefore restricted to subjects with milder asthma. As a result, little is known about the characteristics of airway (Received in original form June 18, 1996 and in revised form January 21, 1997) Supported by grants from Father Sean O'Sullivan Research Foundation, Astra Pharma Inc. and Boehringer Ingleheim (Canada) Ltd. Drs. Marcia Pizzichini and Emilio Pizzichini were supported by a fellowship from C.A.P.E.S., Ministry of Education, Brazil. Correspondence and requests for reprints should be addressed to Dr. F. E. Har -greave, Firestone Regional Chest and Allergy Unit, St. Joseph's Hospital, 50 Charlton Avenue East, Hamilton Ontario, L8N 4A6 Canada. Am J Respir Crit Care Med Vol pp , 1997 inflammation in more severe asthma or severe exacerbations of asthma. Current knowledge is based on postmortem studies and is confounded by poor clinical characterization. The classic pathology of fatal asthma includes the presence of a viscid exudate in the bronchial lumen composed of mucus, eosinophils, and bronchoepithelial cells; thickening of the basement membrane; tissue eosinophilia; thickening of the bronchial smooth muscle layer; and capillary blood vessel dilatation (9). More recently, fatal asthma has been shown to be a heterogeneous condition with the presence of activated eosinophils (EG2+), increased TB lymphocyte ratio, prominent leukocytic infiltration (10), and, in some cases, relatively more neutrophils than eosinophils (11). It is unclear; however, whether the inflammation seen under these circumstances is qualitatively or quantitatively similar to that in non-fatal severe exacerbations of asthma. Another consequence of the failure to measure airway inflammation in severe exacerbations of asthma is a lack of knowledge of the response to anti-inflammatory treatment. In 1983, Baigelman and coworkers (12) studied sputum eosinophils in

2 1502 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL acute excerbations of asthma before and after treatment with prednisone 80 mg for 3 d. They showed that before treatment sputum eosinophils were increased and after treatment they decreased in association with clinical improvement. However, because of the limitations of sputum examination methodology at that time, little attention was paid to this study. In the early 1990s, Corrigan and coworkers (13, 14) reported the presence of activated CD4+ lymphocytes and increased levels of interleukin-5 (IL-5) in the peripheral blood of adults with acute severe asthma, and these were responsive to treatment with corticosteroid to a degree that correlated with the improvement of clinical parameters (14). However, it is unknown to what extent these indices on peripheral blood reflect the inflammatory process in the airways of the lung. A resurgence of interest in sputum examination to directly measure indices of airway inflammation has occurred in the past 8 yr. Sputum induction with an aerosol of hypertonic saline can be performed safely and successfully in mild asthma (4, 15). The method of examination has been improved and evaluated. Examination of sputum separated from saliva for cell and fluid-phase markers has been shown to be an evaluative and discriminative instrument to assess airway inflammation in research (5). We, therefore, sought to examine such sputum in patients with a severe exacerbation of asthma before and after treatment. Our objectives were: (1) to determine the characteristics of the inflammatory cells and their activation markers in sputum; (2) to examine the kinetics of the effects of prednisone treatment on symptoms scores, FEV 1, and sputum inflammatory indices; (3) to compare the indices of inflammation in sputum with those measured indirectly in peripheral blood; and (4) to investigate, in these patients, the safety of sputum induction by a modified procedure. Sputum and blood were examined blind to the clinical details. METHODS Subjects Ten adults with a severe exacerbation of asthma were consecutively recruited from the Firestone Regional Chest and Allergy Clinic and from the Emergency Department of St. Joseph's Hospital between January and November 1995 (Table 1). The diagnosis of asthma had been previously established in seven subjects by symptoms of episodic wheezing, chest tightness, dyspnea, and either methacholine airway hyperresponsiveness (when the FEV I? 70% predicted) in four patients or by an improvement of 15% after salbutamol 200 p.g (when the FEV 1 < 70% predicted or FEV1/VC < 70%) in three other patients. In the three remaining patients, the diagnosis was confirmed during the study by an improvement in the FEV 1 [mean (range)] of 1.9 ( ) L and of 53 (46-64) % (from predicted). A severe exacerbation of asthma was defined by the presence of nocturnal symptoms disturbing sleep (all patients) and/or the need for an inhaled short acting 132-agonist at least 8 puffs/d (six patients) and by an FEV I after bronchodilator of < 60% of predicted. None of the patients had chest radiograph or sputum culture evidence of pulmonary bacterial infection, and none received antibiotics during the study period. One patient had influenza A isolated from a nasopharyngeal swab. Another patient had positive paired serology for respiratory syncytial virus. One patient had mild chronic airflow limitation as indicated by a best FEV I, of 74% during the past year. The study was approved by the hospital research committee and all subjects gave written informed consent. Study Design We performed a descriptive cohort study (Figure 1). Suitable patients were seen before any asthma treatment in the hospital (D o) and then each 24 h at the same time of the day for 3 d (D 1, D2, and D3) and on days 7 and 21 (D 7 and D21). The treatment consisted of prednisone 30 mg/d for 5 d followed by reduction by 5 mg/d to zero by day 10. The dose of prednisone on days 0 to 3 was administered in the research laboratory after sputum induction and blood collection. From D o throughout D25 the patients also received inhaled steroid (budesonide delivered by Turbuhaler or beclomethasone by pressurized inhaler) and inhaled salbutamol by pressurized inhaler as required. Patients who were already on inhaled steroids (? 800 p g/d) were continued on the same dose; those patients who were not or were on a lower dose were begun on budesonide 800 µ/d On D 0,,subject characteristics were documented, spirometry was performed, sputum and blood were collected for inflammatory indices, and a nasopharyngeal swab and sputum were obtained for virus and bacterial culture respectively. On each subsequent day, the questionnaire of symptoms and medication was completed, spirometry was performed, sputum and blood were obtained for inflammatory indices, and the daily diaries were collected. On D o and D21, blood was also taken for serology for virus, Chlamydia pneumoniae, and Mycoplasma pneumoniae. The main outcomes were: symptoms score; postbronchodilator FEV I; sputum total cell count; cell viability; eosinophils; neutrophils; eosinophil cationic protein (ECP); myeloperoxidase (MPO); fibrinogen and IL-5 (only measured on D o and D 7) and blood eosinophils, serum ECP, and IL-5 (only measured on Do and D7). All measurements of blood and sputum were performed blind to the clinical characteristics. Diagnostic Features of Asthma TABLE 1 PATIENT CHARACTERISTICS Before Study During Exacerbation Other Dharacteristics During Exacerbation Age Best FEV J FEV,1 PC,, FEV, FEV, Duration Short Acting Steroid Presumed Patient No. (Sex) Atopy L (%) L (%) (mg/ml) Do D2, Sy Score (days) 132 (puffs)ii Treatment Cause 1 50 (M) Y 3.1 (86) 0.8 (22) (58) 2.7 (71) B 1200 Stop Rx 2 56 (M) Y 2.9 (83) 0.7(20) ,(49) 2.5 (86) Cold 3 25 (F) Y 2.8 (88) 0.9 (28) (50) 2.0 (63) tone Stop Rx 4 33 (F) N 3.2 (106) 0.9 (29) < (47) 3.1 (101) B^ P Unknown 5 50 (F) Y 1.8(80) 0.3(14) - 1.3(59) 1.9(84) , 800 Cold 6 76 (F) N 1.5 (83) 0.3 (17) (50) 1.7 (94) None Stop Rx 7 27 (F) N 3.6 (102) (51) 3.6 (102) B 800 Cold** 8 23 (F) Yt 3.7 (106) (37) 3.5 (100) BDP 400 Cold** 9 16 (F) Y 3.2 (89) 0.3k 1.4 (39) 3.1 (86) None Under Rx 10* 62 (F) yt 2.6 (74) 0.5 (14) (41) 1.7 (44) Cold * = Patient with non eosinophilic and neutrophilic inflammation (see text for details). Atopy means one or more positive allergy skin prick tests. f = ex smokers. $ = best improve. ment in the past year in FEV, after inhaled salbutamol (200 µg) when the FEV 1 < 70% predicted or FEV,NC < 70%. = measured after the study. PC 20 = provocation concentration of methacholine to cause a 20% fall in FEV, if the FEV, >_ 70%. Sy = symptoms score [ranged from 4 (the most severe discomfort) to 36 (asymptomatic)]. B = budesonide in µg/day, BDP = beclomethasone dipropianate in µg/day and P = prednisone 20 mg/day. Rx = treatment. 11 = in 24 hours before admission. ** = confirmed viral infection: Influenzae A (number 7), human respiratory syncytial virus (number 8).

3 Pizzichini, Pizzichini, Clelland, et al.: Sputum Examination 1503 Measurements Descriptive cohort Day Prednison 30mg for 5d then tapered to zero 1 1 Figure 1. Study design. Budesonide 800 to 3200 pg/d Clinical Methods Patient characteristics were documented by questionnaire. During the study period, symptoms (chest tightness, shortness of breath, wheezing, and cough) were graded on a modified Borg scale (16) and recorded in a diary twice daily. Symptoms score ranged from 4 (the most severe discomfort) to 36 (asymptomatic). The use of medications and PEF before and after salbutamol were also recorded in the diary. Spirometry was performed with a PK 131 Morgan Spiroflow dry rolling seal spirometer (Roxon Medi-Tech, Rexdale, Ontario). Spirometry, methacholine inhalation tests and allergy skin tests with 12 common allergen extracts were performed using standard procedures (17-19). Nasopharyngeal swabs were processed by a standard technique of immunofluorescent staining. They were also inoculated onto monolayers of cell lines for isolation of influenza A and B; parainfluenza 1, 2, and 3; adenovirus; and respiratory syncytial viruses (RSV). Human rhinoviruses were sought using reverse transcriptase polymerase chain reaction (PCR) with rhinovirus specific primers, and C. pneumoniae and M. pneumoniae were investigated by PCR using established procedures. Paired samples of serum on D o and D21 were stored at 20 C until examination by complement fixation tests for antibodies to influenza A and B; parainfluenza 1, 2, and 3; adenovirus; RSV; C. pneumoniae; and M. pneumoniae. Sputum Induction Sputum was induced only when it could not be produced spontaneously. The induction procedure described by Pin and colleagues (4) was followed but was made safer by beginning with an aerosol of normal followed by hypertonic saline (3, 4, and 5%) generated by a Fisoneb ultrasonic nebulizer (Canadian Medical Products, Ltd., Markham, Ontario) with an output of 0.87 ml/min and particle size of 5.58 µm aerodynamic mass median diameter. The aerosol was inhaled for 1 or 2 min according to the severity of airflow obstruction, and FEV1 was measured. Patients were asked to rinse the mouth, swallow the water, and blow the nose to minimize contamination with saliva and post-nasal drip. They were instructed to cough sputum into a sterile container whenever they felt that sputum might be present. These procedures were repeated for up to a total of 6 or 7 min of inhalation sequentially with each concentration until a sputum sample was obtained or a fall in the FEV I? 10% occurred. Sputum Examination The appearance of the sputum was recorded as mucoid, mucopurulent, or purulent. Sputum was selected from saliva (20) and processed within 2 h as described by Pizzichini and coworkers (5). Briefly, sputum was treated by adding four volumes of 0.1% dithiothreitol (DTT) (Sputalysin 10%; Calbiochem Corp., San Diego, CA) followed by four volumes of Dulbecco phosphate-buffered saline (D-PBS). The suspension was filtered through a 48-p.m nylon gauze (BBSH Thompson, Scarborough, Ontario), the filtrate was centrifuged at 790 X g for 10 min, and the supernatant was aspirated and stored in Eppendorf tubes at 70 C for later assay. The cell pellet was resuspended in D-PBS, 200 to 600 µl, depending on macroscopic size, and a total cell count of leukocytes and cell viability were determined. The cell suspension was adjusted to 1.0 X 106/ml, placed into cups of Shandon III cytocentrifuge (Shandon Southern Instruments, Sewickley, PA), and two coded cytospins were prepared, air dried, and stained by Wright's stain. At least 200 intact nonsquamous cells were counted on the best stained cytospin. The presence of free granules of eosinophils was reported using an arbitrary scale of few (1), moderate (2), many (3), and excessive (4). Fluid Phase Measurements The concentration of ECP and MPO in pg(l in the thawed supernatant was determined using radioimmunoassay (RIA; Kabi Pharmacia Diagnostics AB, Uppsala, Sweden). Fibrinogen was measured by sandwich ELISA assay using a rabbit anti-human fibrinogen antibody (Dako A080). Interleukin-5 (IL-5, ng/l) was measured by quantitative sandwich enzyme immunoassay (QuantikineTM; R&D Systems, Inc., MN). The limits of detection for the fluid-phase assays were 2.0 µg/l, 8 µg/l, 0.79 µg/l, and 7.8 pg/mi respectively. The results were adjusted for the dilution factor of the procedure (5). Blood Measurements Venous blood was collected into a 5.0 ml EDTA (K 3 Vacutainer BD, Rutherford, New Jersey), and a differential white cell count was obtained using Coutler STKS (Coulter Corp., Hialeah, FL). Serum was collected after blood coagulation for one hour at room temperature. It was centrifuged at 20 C at 1500 rpm for 10 min and stored at 20 C until analysis. The assay used to measure serum ECP and IL-5 was the same as described for sputum. Data Analysis All data were analyzed using the statistical package SPSS for Windows, release 7.0. Results are reported as median and intequartile range (IQR) calculated using weighted averages. Dependent variables with non-normal distribution (eosinophils, ECP, fibrinogen, IL-5, and MPO) were log transformed before analysis. Repeated measures analysis of variance (ANOVA) was used in a model to analyze the effects of two independent variables (time and treatment) on dependent variables (symptoms, FEV 1, and sputum and blood inflammatory indices). Significance was accepted at the level of 95%, and the source of significant variation was identified by the Student-Newman Keuls procedure to adjust the significance for multiple comparisons (21). Spearmann rank correlation coefficients was used to correlate the improvement of different outcomes. Two tailed unpaired t tests were used for comparisons between sputum induction, which lead to a fall in FEV 1 >_ 10% and those with a fall in FEVI < 10%. Regression analysis was used to explain the relation between inhaled salbutamol and % fall in FEVI after sputum induction. RESULTS Inflammatory Indices in Sputum at Presentation Sputum with < 5% of squamous cell contamination was obtained from all patients with the exception of one on one occasion (patient 8 on D21). Twenty-nine samples were spontaneous and 30 induced. The viability of spontaneous and induced sputum was poorer than usual but similar (50.0 [31.0] vs [26.0] %, p = 0.4). Three of the induced samples from one patient (patient 6 on Dt, 7, 21) had so many degenerated cells that it was not possible to make cytospins; however, the supernatants were kept and examined. The main findings at presentation included an eosinophilic inflammatory response with the presence of many free eosinophil granules; low cell viability; and increased levels of fluid phase ECP, fibrinogen; and IL-5 (Table 2). The degree of inflammation between patients was heterogeneous. One patient (number 10) had a noneosinophilic and neutrophilic response, which was maintained throughout the study. Three other patients (numbers 4, 5, and 8) had relatively low levels of ECP and fibrinogen despite the presence of sputum eosinophilia and degranulated eosinophils.

4 1504 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL TABLE 2 BLOOD AND SPUTUM RESULTS AT PRESENTATION Blood Sputum Subject Eo ECP IL-5 V TCC Eo Eo N ECP MPO F IL-5 No. Time (%) (µg/l) (pg/l) Ap (%) (106/m() (%) grn (%) (µg/l) (µg/l) (µg/l) (pg/l) 1 A M A UL M A UL M A UL M M M M M M M ND ND ND M M UL 9 E UL MP A UL MP Median IQR Time is the time of day (M = morning, A = afternoon, E = evening). Definition of abbreviations: Eo = eosinophils; Ap = sputum appearance (M = mucoid, MP = questionably purulent); V = cell viability; TCC = total cell count; Eo grn = free eosinophil granules (1 = few, 2 = moderate, 3 = many, and 4 = excessive); N = neutrophils; F fibrinogen; ND = not done (too few non-squamous cells); UL = under the limit of detection of the assay; IQR = interquartile range. Kinetics of the Effects of Prednisone Treatment on Clinical and Sputum Inflammatory Indices The changes in symptoms, FEV 1 (% predicted), use of inhaled bronchodilator (puffs/day) and sputum inflammatory indices over the 21 d are shown in Table 3 and Figures 2, 3, and 4. These improved in nine of 10 patients. In the nine, the improvement in symptoms was less than the other parameters. The postbronchodilator FEV I (% predicted) increased significantly within 24 h and continued to improve until D 21. The median (minimum and maximum) increase in FEV 1 after treatment was 0.7 ( ) L on Dt and 1.1 ( ) L on D7. In contrast, significant changes in sputum eosinophils, eosinophil granules, and fluid phase ECP were delayed until D2 and D3 and fibrinogen levels until D7. By D21, after prednisone had been discontinued on D 10, the improvement on symptoms and FEV 1 was maintained but there was an increase in sputum eosinophils, ECP, and fibrinogen. We only had enough supernatant to measure IL-5 on D o and D7; on Do, it was present in nine patients, and, on D7, it was reduced to below the lower limit of detection. The improvement in IL-5 (% change on D 7) was strongly correlated with the improvement in eosinophils (rs = 0.96; p < 0.001) and ECP (rs = 0.90; p < 0.001). The improvement in FEV 1 (% from the predicted) was significantly correlated with improvement in sputum eosinophils (rs = 0.65; p = 0.05). Sputum neutrophils and fluid-phase levels of MPO did not change significantly after treatment. The one patient who had no improvement in clinical indices with prednisone treatment differed from the remaining in having sputum neutrophilia without an increase in sputum eosinophils. In this patient, there was also no improvement in any sputum parameter. Subsequently, although sputum was not frankly purulent and did not culture any pathogens, the patient was treated with an antibiotic, and, over 7 d, there was improvement in symptoms and the FEV 1 improved by 0.6 L (17% from predicted). Comparison of Inflammatory Indices in Sputum and Peripheral Blood Peripheral blood, like sputum, showed an eosinophilia and an increase in serum ECP and IL-5. However, the proportion of TABLE 3 KINETICS OF CHANGE IN CLINICAL, BLOOD, AND SPUTUM INDICES AFTER TREATMENT* Clinical Blood Sputum Sy FEV1 132 Eo ECP V TCC Eo Eo N ECP MPO F Day Score (%) (Puffs ) (%) (µg/l) (%) (106/m() (%) gm (%) (µg/l) (µ9/l) (µg/l) (13.8) (12.3) (7.3) (7.6) (35.0) (31.5) (6.8) (16.4) (49.4) (9204) (5008) (10720) * (10.8) (34.0) (4.5) (2.8) (14.8) (26.5) (12.6) (20.5) (51.3) (9210) (4552) (17980) * Ot f t (15.5) (23.1) (4.0) (2.4) (14.0) (30.1) (5.3) (8.2) (30.2) (4252) (4672) (7896) * Ot f # (9.0) (23.2) (4.0) (2.1) (13.3) (33.0) (11.9) (2.3) (31.3) (2598) (4069) (3170) * Ot Oí / (11.0) (25.0) (4.0) (2.1) (18.5) (26.0) (6.2) (1.5) (46.0) (1368) (3621) (4480) Ot 4.9* 23.0í $ * (11.2) (31.0) (6.0) (5.2) (17.2) (30.2) (22.4) (14.5) (32.9) (4640) (6916) (10240) * Median (interquartile range) Abbreviations as in Tables 1 and 2. p Values for comparisons between Da and the respective day: * p = 0.05, t p <0.05, x p < 0.01, and p s 0.001

5 Pizzichini, Pizzichini, Clelland, et al.: Sputum Examination LL a 2 30 E to Pndnhons MNmant Budaonlds 000 to 3200 pyd 21 0J Day ar $u.8 2 m 4 e0 70 gá 60 2 i sb ó 40 d 30 Day Pndnisons trsatmant Budasonlds800to3200 POId 21 0 ay Pn.dnisons treatment 0 Budssonids 800 to 3200 pg1d 21 Figure 2. Individual values of post-bronchodilator FEV, (% predicted), sputum eosinophils (%), and peripheral blood eosinophils (%) before (D o) and after treatment with prednisone (D1,2,3,7,21). Each figure highlights the measurements of the only patient who did not have an eosinophilic exacerbation and did not improve after prednisone treatment (speckled squares). Open circles represents those patients who had sputum induction on D o illustrate the absence of any effect of this on sputum eosinophils on D 1. Stars represent p < cells and the concentration of the serum markers were lower than in sputum; only five of the nine patients who had increased levels of IL-5 in sputum had serum IL-5 above the limits of detection. In contrast to sputum, the blood eosinophils and serum ECP decreased quickly within 24 h of treatment; like sputum, blood eosinophils increased again by D21. Safety of Sputum Induction Only two patientewere able to produce sputum spontaneously on every study day. Sputum induction was required on 30 occasions [Do (4), D 1 (5), D2 (6), D 3 (4), D7 (7), and D21 (4)], and the mean (minimum-maximum) duration of the procedure was 8.4 (2-21) min. The final saline concentration of the aerosol reached in each induction was 0.9% on 7 (23.3%) occasions, 3.0% on 19 (63.3%), 4.0% on 1 (3.3%), and 5.0 on 3 (10.0%) occasions. The procedure was safe. After sputum induction there was a fall in the FEVI ^ 10% (median 13, range 10-42) on 10 occasions and a fall of < 10% (0, 0-9) on 20 occasions. We examined the magnitude of fall in FEV I in relation to various clinical parameters and sputum parameters. The patients in whom inductions caused a fall in the FEVI of 10% after the procedure differed from those with a fall < 10% by having used a higher dose of inhaled (3 2-agonist in the preceding 24 h (8.0 [5.0] versus 4.0 [3.0] puffs/day, p = 0.01), and this was positively correlated with the magnitude of induced fall in FEV 1 (rs = 0.62; p < 0.001). They did not differ in respect to the day of the induction procedure, saline concentration [3.0 (3.0) versus 3.0 (3.0), %], duration of inhalation [9.0 (5.0) versus 7.0 (5.0) min], FEV I before the procedure [70.0 (30.0) versus 72.0 (37.0) % predicted], sputum eosinophils [3.5 (23.90) versus 1.1 (7.1) %], ECP [312.0 (1152.0) versus (1156.0) µg/l], or fibrinogen [ (5240.0) versus (9184.0) µg/l]. DISCUSSION In this study, we investigated the characteristics of airway inflammation in sputum during severe exacerbations of asthma and have followed the kinetics of change after treatment with prednisone. We used a modified sputum induction protocol to increase its safety, when the sputum could not be produced spontaneously. The samples were processed by selecting sputum from saliva to minimize the confounding influence of saliva on the results. The inflammatory responses were characterized by much cell degeneration, relatively poor cell viability, and the presence of many free eosinophil granules. There was a pronounced sputum eosinophilia, elevation of ECP and IL-5, and a marked increase in vascular permeability as measured by sputum fibrinogen. The treatment with prednisone improved the FEVI and blood inflammatory indices within a day, whereas the sputum indices attenuated more slowly but consistently between D2 and D7. The magnitude of improvement in FEV I correlated with the magnitude of fall in sputum eosinophils. The degree of fall in sputum eosinophils and ECP correlated with the degree of fall in sputum IL-5. The longitudinal correlations validate the method of sputum examination used and indicate that the indices measured can be used to study the kinetics of the anti-inflammatory effects of treatment. Several of these observations are new or have not been widely recognized. While lower cell viability, greater degree of cell degeneration, and presence of free eosinophil granules were not observed in a previous study of patients with stable asthma using the same methods (5), they have been seen in the pathology of fatal asthma (9, 10, 22). The delay in the response of sputum eosinophils and ECP to prednisone treatment for 2 d and of sputum fibrinogen for between D 3-D7, has not been reported before. The successful application of the examination of sputum selected from saliva to make sequential measurements reliably is new and so is the safety of sputum induction when this is carried out carefully with a modified protocol. These observations provide new insights into the use of sputum to investigate the pathophysiology and treatment of a severe exacerbation of asthma. This is the first study to demonstrate the responsiveness of cells and soluble markers, measured in sputum selected from saliva, to changes over time. The design or methods cannot be considered to be biased for a number of reasons. First, the

6 1506 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL j EosM ophis ECP ^, 0 25 E E ^ Day PrsdnNont tralnmnt Pndnbaw b~nent 0 aud..mrds 80o t0 32w µgis 21 0 audaomd. aoo to rm0 µya 21 ECP EosMiopIiMi Figure 3. Percentage improvement from Do (median values) of inflammatory indices in sputum (left pane (eosinophils, ECP, and fibrinogen) and peripheral blood (right panen (eosinophils and serum ECP). quality of the observations was increased by incorporating in the design many of the criteria used in randomized controlled trials (RCT), a strategy that has been shown to give results which are nearly identical to those obtained with a RCT (23). Specifically, eligible subjects were required to fulfill standard diagnostic criteria for asthma, and the definition of a severe exacerbation was established beforehand. None of the subjects had purulent sputum, which might suggest a concurrent respiratory bacterial infection. The inception point was the same for all patients and the subsequent measurements were performed at the same time of day and at the same intervals of time. Second, all laboratory measurements were made blind to the subject characteristics. Lastly, various aspects of the method of sputum induction and examination which might influence results have been investigated (20, 24-26); in the main, they are not considered to bias the results. For example, with respect to the use of spontaneous or induced sputum between and within subjects, we have previously shown that sputum induction with same equipment using sequential inhalations of an aerosol of 3, 4, and 5% for a total of 21 min had no effect 400 E CD p=0.006 p= J- -- Day Sputum Serum Figure 4. Effects of prednisone treatment on sputum and serum IL-5. Speckled squares as in Figure 2. Horizontal bars represent median values. Dashed line represents the limits of the detection of the assay (7.8 pg/ml). After prednisone, both sputum and serum IL-5 were undetectable. on the cell counts. It did result in significantly lower fibrinogen levels and a trend for lower ECP which we speculated was due to dilution of the sample with saline inhaled during induction (26). In the present study, however, the latter effects on fluidphase measurements would be expected to be insignificant because the duration of inhalation was much less (mean 8.4 versus 21 min). In the previous study, the cell viability was very good but it was less in spontaneous than induced sputum. In the present study, however, the cell viability was worse but was similar between spontaneous and induced samples throughout the study. This lower cell viability and cell disruption may have been a result of decreased clearance of airway secretions (27) resulting in an older age population of cells or of increased cell killing by cytotoxic products such as ECP (28). There was therefore no evidence to indicate that collection of either spontaneous or induced sputum in the same subject would bias the results. However, as the differential cell counts for eosinophils were made by counting only intact cells, this might have falsely lowered the proportion of eosinophils reported. Together these design and measurements issues lend weight to our findings and decrease the possibility of misinterpreting the results. It could be argued, however, that repeated sputum induction influences the results over 24 h. We could find no full length publication that would lend support to this concern. There is one abstract (29) that describes the effect of sputum induction after 24 hours in 11 healthy subjects. The amount of saline inhaled was greater than in the present study (3, 4, and 5%, each for 10 min). An increase in the proportion of neutrophils (but not in the total cell count or proportion of other cells) was observed. We have addressed this issue in the present study (Figure 2). There was no difference in the proportion of eosinophils and neutrophils between baseline and 24 h. Our results are supported by previous comparisons between paired bronchoalveolar lavages with normal saline within 24 h, which showed no changes in the cell or fluid phase contents (30). The presence of airway eosinophilia (15), raised eosinophil granule proteins (28), and increased airway microvascular permeability (9) are well recognized in exacerbations of asthma. The predominance of eosinophils differs from the observations of Fahy and coworkers (31) who found that neutrophils comprised 75% or more of the sputum cells in 56% of patients with an acute exacerbation of asthma who were seen in the emergency department. The difference in the results of Fahy and colleagues to our own was probably a consequence of the subjects selected with the inclusion of many with a very high total cell count more consistent with bacterial infection. However, this heterogeneity of the inflammatory response in asthma has

7 Pizzichini, Pizzichini, Clelland, et al.: Sputum Examination been observed before (32). In the present study, only one patient had a neutrophilia without eosinophilia. However, among the remainder with sputum eosinophilia and who had a similar degree of airway narrowing, the degree of sputum eosinophilia and fluid-phase markers varied between subjects. The clinical and therapeutic implications of this heterogeneity have not been established. In the present study, it is noteworthy that the treatment with prednisone had no effect on clinicalor inflammatory indices in the subject with neutrophilia. These observations underline the need for objective measurements to discriminate the type and intensity of inflammatory response in patients to better understand exacerbations of asthma symptoms and their treatment. Although corticosteroids are considered to be the most effective treatment of asthma by suppressing airway inflammation, this has not been objectively investigated in a severe exacerbation. In the present study, prednisone treatment in a dose of 30 mgld for 5 d followed by reduction to zero by 010 effectively reduced the various inflammatory measurements by 0 7, Symptoms, FEVh blood eosinophils, and ECP improved quickly while the sputum measurements were delayed. This suggests that the clinical and blood measurements do not accurately reflect the airway inflammatory response to prednisone. The early improvement of bronchoconstriction has also been observed in otherstudies (33). Whether this early improvement in the present study was due to a reduction of mediators of constriction and/or other aspects of airway inflammation requires further investigations. Glucocorticosteroids suppress cytokine gene transcription factors activated by the inflammatory process (34). Our results showing attenuation in the proportion of eosinophils, free eosinophil granules and ECP and strong correlations between these findings and the suppression of IL-5 production after treatment support this notion and are similar to another study using bronchial biopsies to assess the effects of prednisone treatment in mild asthmatics (35). Corticosteroids may also decrease the leakage of the airway microvasculature. The observation that fibrinogen in sputum supernatant progressively decreased after a week of prednisone treatment are consistent with animal studies showing that dexamethasone markedly inhibits plasma exudation in the airways of sensitized rats challenged with allergen or platelet activation factor (PAF) (36). The reason for the deterioration in sputum and blood indices on 0 21 was not investigated. It could have been a result of poor compliance or an inadequate dose of inhaled steroid. In this study, we performed sputum induction carefully. It should be noted that the Fisoneb has a relatively low output for an ultrasonic nebulizer, and that we began inhalations with an aerosol of normal saline for short periods of 1 min followed by up to three periods of 2 min before progressing to hypertonic saline; we then measured the FEV1 after each 1 or 2 min of inhalation and we stopped the inhalations as soon as enough sputum was obtained for examination or the FEV 1 had fallen by ~ 10% from baseline. Induced sputum was easily obtained with :5 8 min of inhalation in almost half of the procedures. The FEV 1 fell by ~ 10% in 10 of the 30 inductions, and this was associated with the use of higher doses of inhaled salbutamol in the 24 h before presentation which is recognized to reduce the protective effect against bronchoconstrictive stimuli of which an inhaled aerosol of saline is one. The greatest fall in FEV 1 occurred in a patient with a post-salbutamol FEV 1 of 93% predicted who had been treated with prednisone for 24 h and had used 13 puffs of salbutamol on the day before. After inhalation of hypertonic saline 3% for a total of 3 min, the FEV 1 fell by 42% (1.3 L) and did not return to baseline for 3 h despite treatment with additional 1507 salbutamol. The observations in this patient indicate that the baseline FEV 1 by itself will not necessarily predict the bronchoconstrictive effect of inhaled saline, and emphasizes the care which must be taken in applying the procedure. We conclude from the results of this study that the examination of spontaneous or induced sputum selected from saliva can be used successfully to examine and follow the effects of treatment in a severe exacerbation of asthma. The effects of anti-inflammatory treatment are not necessarily reflected by symptoms, FEV 1 or blood eosinophils or ECP. During the study the only subject who did not improve objectively with prednisone had no sputum eosinophilia. It is reasonable to speculate that measurements of sputum eosinophils will be useful in predicting the response to added steroid treatment. Acknowledgments: The authors thank the patients who agreed to participate in this study, the physicians and nursesof the Emergency Department of StJoseph'sHospital and the FirestoneRegional Chest and Allergy Unit for help with recruiting the subjects, Sharon Weston for helping with cell counts, Susan Evans for performing the fluid-phase measurements, Maureen Thomas for serology and immunofluorescence for virus, Silvia Chong for searching for rhinovirus, and Pharmacia Diagnostics AB, Uppsala, Sweden for providing the ECP kits. References 1. Laitinen, L. A, M. Heino, A Laitinen, T. Hava, and T. Haahtela Damage of the airway epithelium and bronchial reactivity in patients with asthma. Am. Rev. Respir. Dis. 131:59~ Beasley, R, R R Roche, J. 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