Cetrimide

Study objectives: To study the correlations between sputum elastase output with clinical and sputum inflammatory and microbial factors in steady-state bronchiectasis.

Design: Prospective recruitment of patients with bronchiectasis (17 women; 48.5 [+ or -] 16.5 years old; [FEV.sub.1]/FVC, 1.3 [+ or -] 0.6/2.1 [+ or -] 0.9) for assessment of 24-h sputum output of elastase, bacteria, leukocytes, interleukin (IL)-1[Beta], IL-8, tumor necrosis factor-[Alpha], and leukotriene [B.sub.4]. Clinical variables assessed concomitantly included 24-h sputum volume, lung spirometry, number of lung lobes affected by bronchiectasis, and exacerbation frequency.

Setting: Consecutive recruitment of outpatients (n = 30) in steady-state bronchiectasis.

Measurements and results: Twenty-four-hour sputum elastase output correlated with 24-h sputum volume (r = 0.79, p = 0.0001); number of bronchiectatic lung lobes (r = 0.54, p = 0.0026); percent predicted [FEV.sub.1] (r = -0.48, p = 0.0068); percent predicted FVC (r = -0.49, p = 0.001); and leukocyte output (r = 0.75, p = 0.0001). There was no correlation between the sputum output of bacteria with either inflammatory or enzymatic factors (p [is greater than] 0.05).

Conclusion: Our data highlight the importance of elastase and the possibility of independent roles for enzymatic, inflammatory, and microbial components in the pathogenesis of bronchiectasis. Further research on novel therapy targeting each of these components should be pursued.

(CHEST 2000; 117:420-426)

Key words: bronchiectasis; elastase; interleukin; leukotriene; sputum

Abbreviations: CF = cystic fibrosis; IL = interleukin; [LTB.sub.4] = leukotriene [B.sub.4]; TNF = tumor necrosis factor

Bronchiectasis is defined pathologically as permanent dilatation of the bronchi. Affected patients suffer from chronic sputum production, recurrent exacerbations, and sometimes progressive lung destruction. The pathogenesis of bronchiectasis is not well understood, but recent studies have identified infective, inflammatory, and also enzymatic elements that interact with each other leading to a vicious cycle of tracheobronchial destruction.[1] The pathogenetic role of common pathogens, such as Pseudomonas aeruginosa, Streptococcus pneumoniae, and Haemophilus influenzae, has received considerable attention recently.[2-4] Extensive recruitment of neutrophils occurs in the bronchiectatic airways,[1,5,6] which is mediated by proinflammatory mediators, including interleukin (IL)-1, IL-8, tumor necrosis factor (TNF)-[Alpha], and leukotriene [B.sub.4] ([LTB.sub.4]).[7-11] Activated neutrophils release intracellular elastase in the bronchiectatic airways, which slows ciliary beating and disrupts respiratory mucosa in vitro.[12,13] Elastase might therefore play an important role in the pathogenesis of bronchiectasis although this has not been investigated previously. Inasmuch as there is no "gold standard" for measuring disease severity or activity, researchers have adopted some clinical and laboratory variables as disease markers in bronchiectasis. These include spirometry, sputum volume measurement, exacerbation frequency, and sputum concentrations of proinflammatory mediators.[8,11,14-19] Because little is known about the relationship between sputum elastase and these variables, we have performed this prospective study to evaluate these correlations in steady-state bronchiectasis.

MATERIALS AND METHODS

Study Design and Patient Recruitment

Patients with proven bronchiectasis diagnosed by high-resolution CT were recruited and gave written informed consent. Inclusion criteria included the absence of asthma or other unstable systemic diseases, no alteration in medication and dose for at least 3 months, and steady-state bronchiectasis. The latter was defined as the presence of [is less than] 20% alteration in 24-h sputum volume, [FEV.sub.1], and FVC, and absence of changes in respiratory symptoms for 3 consecutive weeks. The study protocol had approval from the institutional ethics committee. Each patient entered a baseline period (3 consecutive weekly visits) to ensure that she or he was in steady-state bronchiectasis before being further assessed for clinical and laboratory variables by a research physician and a technician blinded to the study protocol.

Variables Assessed

At each visit, the patients were directly asked about the presence of respiratory symptoms (cough, dyspnea, hemoptysis, sputum production, chest pain, and wheezing) and were examined physically. Clinical assessment included the determination of the exacerbation frequency, spirometry, and the number of bronchiectatic lung lobes for each patient. Exacerbation frequency was defined as the number of exacerbations that had occurred in the preceding 12 months. This was determined by meticulous history taking and review of clinical charts. Occurrence and severity of respiratory symptoms, including cough, dyspnea, hemoptysis, increased sputum purulence or volume, and chest pain, were assessed for each patient. An exacerbation was defined as subjective and persistent ([is greater than or equal to] 24 h) deterioration in at least three respiratory symptoms, with or without fever ([is greater than or equal to] 37.5 [degrees] C), radiographic deterioration, systemic disturbances, or deterioration in percussion note or auscultatory findings in the chest.[14] Spirometry ([FEV.sub.1] and FVC), expressed as percent predicted, was measured between 10:00 AM and 12:00 PM with a SensorMedics 2200 (SensorMedics; Yorba Linda, CA) package. Thoracic high-resolution CT was performed, within 12 months of the study, using a General Electric Hispeed Advantage Scanner (Milwaukee, WI) to perform standard 1-mm-thick sections at 10-mm intervals in the supine position. The number of lung lobes (including lingula) affected by bronchiectasis, as evident by the bronchial segment or subsegment being larger than the accompanying artery,[20] was determined for each patient. Laboratory assessment included 24-h sputum volume; sputum leukocyte density (per milliliter); sputum total bacterial densities (colony forming units per milliliter); and sputum (sol phase) concentrations of IL-1[Alpha], IL-8, and TNF-[Alpha], [LTB.sub.4], and elastase.

Measurement of Sputum Sol Elastase

Fresh sputum was stored at -70 [degrees] C within 15 min of collection until ultracentrifugation (100,000g for 30 min at 4 [degrees] C) to obtain the sol phase, which was used for determination of elastase activity (concentration). Briefly, 5 [micro]L of sputum sol was added to a chromogenic peptide substrate succinyl-L-alanyl-L-alanine-[Rho]-nitroanilide (Sigma; Dorset, UK), and the rate of change of optical density was determined at 410 nm by using a spectrophotometer.[21] This rate was compared with a standard curve for the rate of change in optical density, which was obtained from incubating known concentrations of elastase solutions (Sigma) with the same chromogen. The rate of change in optical density was converted into elastase activity (concentration) and expressed in units per milliliter. The elastase concentration was determined in triplicate, and the mean was determined for each patient.

Assessment of Sputum Physical Characteristics

The volume of a 24-h sputum specimen was determined as the mean of a 3-consecutive-day collection (9:00 AM to 9:00 AM) as described previously.[14] Briefly, 24-h sputum collection was made by the patients at home in clear sterile plastic (60 mL) pots and stored at 4 [degrees] C. Patients were trained to completely empty the contents of their mouth before expectoration. Contamination of sputum with visible saliva and food debris was infrequently encountered after the baseline visits. The volume of a 24-h sputum specimen was determined to the nearest 0.5 mL.[14] Patients received chest physiotherapy (at least 15 min of expectoration-aiding maneuvers and until no further sputum was obtained) on arrival at the clinic. Fresh sputum was then collected by the research physician in sterile clear plastic pots between 10:00 AM and 12:00 PM after thorough mouth emptying, and within 1 h of physiotherapy in the semireclined position. Sputum leukocyte density, performed on five randomly selected aliquots of a fresh specimen, was assessed within 2 h of collection by the same technician using light microscopy and a hemocytometer.[14]

Determination of Sputum Bacterial Densities

Standard microbiological procedures were used to identify all the sputum bacteria and classify them into pathogens (P aeruginosa, H influenzae, S pneumoniae, Staphylococcus aureus, Moraxella catarrhalis, and Mycobacteria species) or nonpathogenic bacteria (Neisseria species, [Alpha]-hemolytic streptococci, diphtheroids, and coagulase-negative staphylococci). The following enriched and selective media were used for determining the bacterial density (colony forming units per milliliter) in sputum: blood agar (Oxoid CM271 [Oxoid; Basingstoke, UK] supplemented with 5% defibrinated horse blood), chocolate agar supplemented with 18.9 U/mL bacitracin (Sigma; St. Louis, MO), mannitol salt agar (Oxoid CM85), and cetrimide-nalidixic acid agar (Oxoid CM559 and SR102). Fresh sputum was homogenized by using SPUTASOL (Oxoid SR089A) and inoculated onto the media with a 10-[micro]L standard plastic loop to determine the microbial densities of various bacteria. Incubation was performed for up to 4 days at 37[degrees]C in 5% [CO.sub.2], and the dilution that gave 30 to 300 cfu after overnight incubation was counted.[14]

Measurement of Sputum Sol Proinflammatory Cytokine and [LTB.sub.4] Concentrations

Sputum sol was obtained, as described above, for determination of cytokine and [LTB.sub.4] concentrations by using enzyme-linked immunosorbent assay. Samples were added to a 96-well plate (R&D Systems; Minneapolis, MN) coated with monoclonal antibody against one of the cytokines or [LTB.sub.4] and incubated for 2 h at room temperature. After this, the samples were removed and washed three times with buffer, and an enzyme-linked antibody specific for a particular cytokine or [LTB.sub.4] was added to each well and incubated at room temperature for 2 h. After a final wash to remove all unbound antibody, a substrate solution was added to each well and incubated for 20 min before the reaction was terminated by adding a stop solution. The optical density was determined by using a plate reader at 450 nm to determine the concentration of the cytokines or [LTB.sub.4] in the sputum, and the mean concentration for each sample was obtained from the triplicate measurements.

Data Analysis and Statistical Methods

The physiologic measurements and cytokine concentrations were log-normally distributed, whereas the other microbial variables were highly skewed. The relationships between sputum variables, sputum biochemistry, and clinical variables were examined using Spearman rank correlation. For each patient, the 24-h sputum output of bacteria was calculated, as was the product of the 24-h sputum volume and the sputum bacterial density. The 24-h sputum outputs of the proinflammatory mediators and elastase were calculated likewise for each patient. The effects of sputum pathogens on various clinical, biochemical, and sputum variables were initially examined using analysis of variance. Because of small number and the lack of difference among various pathogen groups other than Pseudomonas, sputum pathogens were reclassified as Pseudomonas and non-Pseudomonas. Comparison between the latter groups was made using unpaired Student's t test after natural logarithmic transformation of the data. All statistical analyses were performed using Statistical Analysis System software package (Version 6.12; SAS Institute; Gary, NC). A p value [is less than] 0.05 was taken as indicative of statistical significance.

RESULTS

Patient Demography and Clinical Details

Between December 1996 and February 1998, 30 non-cystic fibrosis (CF) patients were consecutively recruited. Their clinical features are shown in Table 1. The majority of patients were never- or ex-smokers. All the patients suffered from bronchiectasis of the cylindrical type on high-resolution CT asessment. The cause of bronchiectasis was classified as idiopathic, posttuberculous, Kartagener's syndrome, and diffuse panbronchiolitis in 25, 1, 3, and 1 patients, respectively.[5] One bone marrow transplant recipient (male, age 33 years) was on maintenance therapy of prednisolone and cyclosporin A. Another patient (female, age 79 years) was on regular therapy of prednisolone and azathioprine for stable idiopathic thrombocytopenic purpura. Each patient received twice daily expectoration-aiding chest physiotherapy at home, which was provided either by the spouse or another designated family member. The cohort of patients had a mean ([+ or -] SD) of 3.6 [+ or -] 3.2 exacerbations in thee preceding 12 months. Twenty-one, 7, and 2 patients displayed obstructive, restrictive, and normal spirometry, respectively.

Table 1--Clinical Characteristics of Patients in Steady-State Bronchiectasis(*)

(*) Data are presented as No. or as mean [+ or -] SD; p values were derived from comparison between the P aeruginosa-infected and noninfected subgroups of patients.

Sputum Physical, Microbial, Enzymatic, and Proinflammatory Mediator Profile Assessment

The clinical and sputum variables were log-normally distributed and were subclassified according to the status of P aeruginosa infection (Tables 2 and 3). Bacteria isolated from the sputum were P aeruginosa (n = 22), H influenzae (n = 4), Mycobacterium chelonae (n - 1), S aureus (n = 1), and S pneumoniae (n = 1). No pathogen was isolated in one case. Patients who had P aeruginosa in their sputum had a significantly higher 24-h sputum output of leukocytes, bacteria, TNF-[Alpha], and [LTB.sub.4] (p [is less than] 0.05), but not elastase (p [is greater than] 0.05), than their counterparts (Table 3).

Table 2--Clinical and Sputum Factors in 30 Patients With Steady-State Bronchiectasis(*)

(*) Data shown have been log-transformed for clarity; M = male; F - female.

Table 3--Clinical and Sputum Variables in 30 Patients With Steady-State Bronchiectasis, Which Are Also Classified According to the Status of P aeruginosa Infection(*)

(*) Data shown have been log-transformed for clarity and are presented as mean ([+ or -] SD). Anti-log of the group values would yield the geometric means and confidence intervals. The p values were obtained by comparing variables between Pseudomonas- and non-Pseudomonas-infected group using unpaired Student's t test on log-transformed values.

Intercorrelation of Sputum Variables

The intercorrelations among various sputum variables are shown in Table 4. Elastase correlated with 24-h sputum volume and sputum leukocyte density (p [is less than] 0.05). Among the sputum proinflammatory mediators, IL-1[Beta], IL-8, and TNF-[Alpha] significantly correlated with each other and with leukocyte density (p [is less than] 0.05, data not shown).

Table 4--Correlation Analysis for Sputum Variables From 30 Patients With Steady-State Bronchiectasis(*)

(*) Data shown are Spearman rank correlation coefficients (p values).

Intercorrelation Between Sputum Variables and Clinical Disease Markers

The intercorrelations among various sputum variables and clinical disease markers, namely number of bronchiectatic lung lobes, exacerbation frequency, [FEV.sub.1] (percent predicted), and FVC (percent predicted), are shown in Table 5. Sputum output of elastase correlated with the number of bronchiectatic lung lobes but not exacerbation frequency, and inversely with spirometry (Fig 1). The number of bronchiectatic lung lobes also significantly correlated with 24-h sputum leukocyte output. Both 24-h sputum volume and 24-h sputum leukocyte output correlated inversely with [FEV.sub.1] (percent predicted) and FVC (percent predicted). Among the indices of disease severity (data not shown), the number of bronchiectatic lung lobes correlated with exacerbation frequency (r = 0.40, p = 0.03), and inversely with [FEV.sub.1] (r = -0.44, p = 0.02) and FVC (r=-0.42, p=0.02). Exacerbation frequency, however, had no correlation with [FEV.sub.1] (r = -0.08, p = 0.69) or FVC (r = -0.14, p = 0.06).

[Figure 1 ILLUSTRATION OMITTED]

Table 5--Relationship Between Clinical Markers of Disease Severity and Sputum Variables in 30 Patients With Steady-State Bronchiectasis(*)

(*) Data shown are Spearman rank correlation coefficients (p values).

([dagger]) Values with p [is less than] 0.05.

DISCUSSION

This study was performed to evaluate the intercorrelation between sputum elastase output and other disease factors in a cohort of 30 patients with steady-state bronchiectasis. Sputum elastase output correlated with 24-h sputum volume, number of bronchiectasis lung lobes, spirometry (negatively), and 24-h sputum output of leukocytes, IL-1[Beta], and TNF-[Alpha]. Sputum elastase output therefore appears to correlate with disease activity, severity, and inflammatory markers in steady-state bronchiectasis. There were significant positive correlations among the 24-h sputum outputs of inflammatory markers (including the cytokines IL-1[Beta], IL-8, and TNF-[Alpha] and leukocytes. An inverse relationship between structural and functional markers was found in that the number of bronchiectatic lung lobes inversely correlated with [FEV.sub.1] (percent predicted) and FVC (percent predicted). Not withstanding the relatively small sample size and the predominance of P aeruginosa infection (73.3% of patients), there was no difference in the correlation patterns between the P aeruginosa-infected patients and their counterparts. Our data showed no correlation between the number of bronchiectatic lung lobes and 24-h sputum volume. This might have been because of the crude nature of only assessing the number of bronchiectatic lobes, rather than the volume or surface area of bronchi that were affected by bronchiectasis. In addition, the presence of bronchiectasis in a lung lobe does not necessarily indicate an underlying active disease process. Very importantly, our data show no correlation between sputum bacterial output and either inflammatory or enzymatic outputs.

Extensive airway infiltration with neutrophils occurs in bronchiectasis, which is mediated by proinflammatory mediators, particularly IL-1[Beta], IL-8, TNF-[Alpha], and [LTB.sub.4].[7,9-11,22] Most patients with non-CF bronchiectasis suffer from airway colonization with H influenzae and S pneumoniae initially, which is followed by chronic colonization by P aeruginosa. Exotoxins produced by P aeruginosa cause ultrastructural damage,[3,4] slowing of ciliary beating,[2] upregulation of respiratory mucus secretion,[23] and induction of TNF-[Alpha], [LTB.sub.4], and IL-8 release from respiratory mucosa in vitro.[24,25] However, our data did not show any in vivo correlation between sputum output of bacteria and proinflammatory mediators (Tables 4, 5). This lack of correlation between sputum proinflammatory mediators, including IL-1[Beta], IL-8, [LTB.sub.4], and TNF-[Alpha], and lung function variables and exacerbations in bronchiectasis has been reported previously.[18,26,27] The presence of severe pulmonary inflammation without any evidence of infection has also been reported in CF lungs.[28] Our data, along with the results from previous studies,[18,26-28] therefore suggest that inflammation in bronchiectasis could be partly independent of the infective process.

Neutrophils recruited into the airways release elastase, hydrogen peroxide, and reactive oxygen radicals, which are toxic to respiratory mucosa.[29] Elastase digests elastin, basement membrane collagen, and proteoglycan.[13] Elastase in the airways, irrespective of its neutrophil or P aeruginosa origin, causes slowing of ciliary beating,[12] extrusion of epithelial cells,[12] and induction of airway mucus production.[30] Sputum elastase concentration has previously been reported to correlate positively with radiographic severity[31] and negatively with lung function in CF and non-CF bronchiectasis.[19] Our data show a correlation of elastase output with 24-h sputum volume and sputum leukocyte output, but not P aeruginosa output. This strongly suggests that most of the sputum elastase were released by neutrophils rather than P aeruginosa.

There is no effective disease-modifying treatment for bronchiectasis. The use of maintenance antibiotics such as nebulized aminoglycosides and judicial early use of potent antibiotics are undoubtedly effective but only treat infection. Prolonged high-dose antibiotic[32] and systemic steroid therapy[33] have failed to produce significant clinical improvement. Our results suggest that the enzymatic, inflammatory, and infective pathogenic elements could be individually treated. Nebulized [Alpha]-antitrypsin reduces lung elastase concentration in CF[34] and might be a potentially useful antielastase treatment. Bronchial epithelial cell cytokine products could also be potential targets for anticytokine therapy. For example, aerosolized IL-1 receptor antagonist reduces TNF-[Alpha] bioavailability in guinea pigs,[35] TNF-[Alpha] and IL-1 receptors reduce bacterial endotoxin-induced neutrophil recruitment to rat lungs,[36] and [F([ab.sup.1]).sub.2] fragments of IL-8 monoclonal antibody reduce sputum chemotactic activity.[11] Our results suggest that novel combinations of these antibacterial, anti-inflammatory, and antienzymatic modes of therapies could be useful.

ACKNOWLEDGMENT: The authors thank Dr. Ian Lauder for expert statistical advice. We are grateful to the patients who participated in this study and the support from Dr. C.S. Ho, Ms. Shelley Chan, and Mr. Raymond Leung for technical assistance.

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(*) From the University Departments of Medicine (Drs. Tsang, Zheng, J.C.M. Ho, and Lam), Microbiology (Dr. P. Ho), Diagnostic Radiology (Dr. Ooi), and Paediatrics (Dr. Chan), The University of Hong Kong, Queen Mary Hospital, Pokfulam, Hong Kong SAR, China.

Manuscript received February 11, 1999; revision accepted July 15, 1999.

Correspondence to: Kenneth W.T. Tsang MD (Hons), FCCP, University Department of Medicine, Queen Mary Hospital, Pokfulam, Hong Kong SAR, China; e-mail: kwttsang@hkucc.hku.hk

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