INTRODUCTION

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Keywords: bronchiectasis; bronchoalveolar lavage; cytokines; bacterial colonization

Bronchiectasis is a chronic pulmonary disease characterized by an irreversible dilatation of the bronchi. The current view of the pathogenesis of bronchiectasis considers initial colonization of the lower respiratory tract by different microorganisms as the first step leading to an inflammatory response characterized by neutrophil migration within the airways and secondary secretion of a variety of tissue-damaging oxidants and enzymes such as neutrophil elastase and myeloperoxidase (1). Persistence of microorganisms in the airways because of impairment in mucus clearance may lead to a vicious circle of events characterized by chronic bacterial colonization, persistent inflammatory reaction, and progressive tissue damage.

Recent studies in patients with cystic fibrosis have shown active inflammation in the airways in the early phases of the disease even in the absence of colonizing microorganisms (4). In these studies, further evaluation of the components of the inflammatory reaction demonstrated low levels of certain cytokines with anti-inflammatory functions, suggesting that an imbalance in the immunologic mechanisms that control the inflammatory process in the airways may influence the evolution of cystic fibrosis. In this sense, early therapeutic intervention aimed at both diminishing the premature inflammation and replacing defective immunomediators may potentially hinder progression of the disease.

Since the intimal mechanisms leading to the development of bronchiectasis in patients with cystic fibrosis may not necessarily prevail in other forms of bronchiectasis, it would be interesting to investigate the following aspects in patients with non-cystic-fibrosis bronchiectasis: (1) the characteristics of bronchial inflammatory reaction, (2) the relationship between airway inflammation and presence of microorganisms with potential pathogenicity (MPP), and (3) the local or systemic nature of the inflammatory response (compartmentalization).

The present study evaluated bronchial and systemic inflammatory response and its relationship to bacterial colonization in patients with bronchiectasis in a stable clinical condition. We employed bronchoalveolar lavage (BAL) as the gold standard technique since this bronchoscopic procedure allows the simultaneous evaluation of: (1) the bronchial inflammatory process directly in the local milieu, and (2) the bacterial colonization pattern in lung segments in which bronchiectasis is located.

	METHODS

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Patients

We prospectively studied 49 patients with bronchiectasis in a stable clinical situation diagnosed by clinical and high resolution chest CT scan criteria and nine nonsmoking subjects without respiratory disease.

Study Protocol

The Ethical Committee of the Hospital Clinic approved the study protocol. Informed consent was obtained from all the patients.

An HRCT scan of the thorax was performed in all the patients within the 3 mo previous to the bronchoscopic examination. The extent and type of bronchiectasis were evaluated in each case as described by Reiff and colleagues (9). On the day of the study, a forced spirometry (Survey III plus; Warren E. Collins, Braintree, MA) was performed before bronchoscopy. Upper airway anesthesia for bronchoscopic examination was achieved by nebulization with 8 ml of lignocaine 5% through a buccal clip for 15 min. The bronchoscope (Olympus BF 30; Olympus Corp, New Hyde Park, NY) was passed transnasally and BAL was performed in the lobe with more involvement by bronchiectasis according to the chest CT scan. The total volume of the recovered fluid was mixed and divided into two different aliquots for cytologic examination/cytokine measurements and quantitative bacterial cultures.

Cytologic and Biochemic Measurements

Total and differential BAL cell counts were determined as described (10). The concentration of elastase and myeloperoxidase (MPO) and the cytokines TNF-, IL-1, IL-6, IL-8, and IL-10 were measured in BAL fluid (BALF) supernatant. Similarly, the same set of cytokines were also assessed in blood samples. Elastase was analyzed using an immunoassay technique. MPO was analyzed using a spectophotometric technique (11).

Cytokines were measured using an enzyme-linked immunosorbent assay (ELISA) based on the quantitative immunometric sandwich enzyme immunoassay technique in a microtiter plate (Enzyme Amplified Sensitivity immunoassay [EASIA]) (EASIA-reader; Medgenix Diagnostics, Fleurus, Belgium (IL-6, TNF-, IL-1) and PerSeptive (Genzyme Corp., Framingham, MA) (IL-8 and IL-10) were used (12). The following values are regarded as upper limits for cytokine serum concentrations in normal control subjects in our laboratory: IL-6 = 5 pg × ml¹, TNF- = 20 pg × ml¹, and IL-1 = 15 pg × ml¹.

Microbiologic Evaluation

Respiratory samples were Gram-stained and homogenized. Undiluted as well as serial-diluted secretions (10¹, 10², 10³) were plated on blood, chocolate, Wilkens-Chalgren, and Sabouraud agar. Cultures were evaluated for growth after 24 and 48 h. Identification of microorganisms was performed according to standard methods (13). Results are expressed in colony-forming units per milliliter (cfu/ml).

Bacterial agents were classified into microorganisms with potential pathogenicity (MPP) or microorganisms with nonpotential pathogenicity, as described elsewhere (14). Only pathogens with counts of  10³ cfu × ml¹ in BALF were regarded as significant.

Statistical Analysis

Data are presented as median and range because of the nonparametric distribution of the data. Differences between the three groups were determined using a nonparametric one-way analysis of variance (Kruskal-Wallis). Individual differences between groups were determined using the Mann-Whitney U test. The chi-square test or Fisher's exact test were used for comparison of categorical variables when appropriate. All reported p values are two-tailed. The level of significance was established as 5% for all analyses. Correlations were calculated using Spearman's rank test.

	RESULTS

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Patients

The main clinical characteristics of the patients enrolled in the study are summarized in Table 1. Women constituted 65% of the patients evaluated. Only three patients (6%) were current smokers and eight (16%) were ex-smokers. Twenty-nine patients (59%) had chronic expectoration. The time of potential pulmonary insult causing bronchiectasis was before the age of 14 yr in 28 subjects (57%). Although, the mean spirometric values were almost within the range of normality, the values of both FVC and FEV₁ were significantly lower than those observed in control subjects. Twenty-four patients (49%) showed airflow obstruction with a FEV1/FVC < 70% and FEV₁ < 80% predicted. Thirty-seven patients (76%) had cylindrical bronchiectasis according to the HRCT scan, eight (16%) cystic bronchiectasis and four (8%) varicose bronchiectasis. Twenty patients (60%) had inflammatory infiltrates associated with bronchiectasis. The mean CT score was 38.8 ± 21%.

Bronchial Markers of Inflammation

The amount of BALF recovered was higher in control subjects than in patients (46 ± 9 ml versus 30 ± 12 ml, p < 0.001). Patients with bronchiectasis showed a significant increase in the percentage of BALF neutrophils and in the concentration of both elastase and MPO compared with control subjects (table 2). There was a good correlation between the percentage of neutrophils in BAL and the concentration of elastase (r = 0.67, p < 0.001) and MPO (r = 0.74, p < 0.001). Patients with bronchiectasis had increased levels of TNF-, IL-8, and IL-6 compared with control subjects. Although the median concentration of IL-10 was higher in patients with bronchiectasis than in control subjects, the difference did not reach statistical significance. There were significant correlations between the concentration of IL-8 and both, the percentage of neutrophils (r = 0.80, p < 0.001) and the concentration of elastase (r = 0.69, p < 0.001) in BALF. There were also fair correlations between the level of TNF- and both, the percentage of neutrophils (r = 0.6, p < 0.001) and the concentration of elastase (r = 0.49, p < 0.05) in BALF.

Microbiologic Results

BAL revealed the presence of MPP above the established threshold ( 10³ cfu/ml) in 22 of 45 patients (48%). In four patients the quality of BAL samples precluded microbiologic analysis. The most common MPP isolated were: 11 nontypable Haemophilus influenzae, eight Pseudomonas spp., one Nocardia asteriodes, one Proteus mirabilis, one Alcalygenes xilosoxidans, one Escherichia coli and one Streptococcus pneumoniae. In two patients, two different microorganisms grew simultaneously on culture above the established threshold (S. pneumoniae plus H. influenzae and H. influenzae plus P. aeruginosa). The microorganisms with nonpotential pathogenicity recovered in this study were as follows: seven Streptococcus viridans, two Neisseria spp., three Corynebacterium spp., and one coagulase negative Staphylococcus.

Relationship between Bacterial Colonization and Indices of Airway Inflammation

We reasoned that the bronchial inflammatory reaction observed in patients with bronchiectasis may be modulated by the presence of MPP colonizing the lower respiratory tract. Thus, in order to assess the effect of colonization on markers of inflammation, patients were grouped according to the presence or absence of MPP in BAL cultures. We observed that patients with bronchiectasis and negative cultures for MPP in the BALF had a more intense inflammatory reaction than did control subjects, with a higher percentage of neutrophils and higher concentrations of IL-8 and IL-6 in BALF (Table 3).

On comparison of the airway inflammatory characteristics between patients with bronchiectasis colonized by MPP (n = 22) and non-colonized patients with bronchiectasis (n = 23), we observed that the group of patients with MPP in the airways had a higher BAL neutrophil count, higher BALF concentrations of both elastase and MPO and higher BALF levels of TNF- and IL-8. The levels of IL-1, IL-6 and IL-10 were also higher in patients with MPP in the airways, although the differences did not reach statistical significance (Table 3).

Interestingly, we observed a clear relationship between the bacterial load in the airways, expressed as colony-forming units and the percentage of neutrophils in BALF and the concentration of both IL-1B and IL-8 (Figure 1).

View larger version (15K): [in this window] [in a new window]   View larger version (12K): [in this window] [in a new window]   View larger version (14K): [in this window] [in a new window]   View larger version (13K): [in this window] [in a new window]   Figure 1.   Relationship between airway bacterial load (cfu/ ml) and the % of neutrophils and TNF-, IL-1, IL-8 concentrations in BALF. Asterisks indicate significant differences when compared with normal flora samples (*p < 0.01). Bars and error bars represent mean and standard deviation. Normal flora = mixed normal flora without viable bacteria or with less than 103 cfu/ml of normal mixed oropharyngeal commensal organisms.

Association between Airway Inflammation and Lung Function

There was no apparent correlation between the different inflammatory parameters evaluated in the BALF and the percentage of both FEV₁ and FVC. However, when we divided patients with bronchiectasis into those with normal lung function (n = 24) and those with moderate obstruction (FEV₁ < 65% predicted) (n = 11), we observed that the latter group had higher BALF levels of IL-1 (185 ± 81 versus 115 ± 59 pg/ml, p < 0.05), TNF- (41 ± 56 versus 11 ± 23 pg/ml, p < 0.05), and IL-10 (4.6 ± 7.3 versus 1.1 ± 2.1 pg/ml, p < 0.05) suggesting that patients with more advanced disease also have more intense bronchial inflammation.

Systemic Inflammatory Response

With the aim of knowing whether the inflammatory response of the airways can be accurately assessed systemically, we determined the concentrations of different inflammatory mediators in plasma. As a group, patients with bronchiectasis showed a slight systemic inflammatory response with most of the plasma mediators (except TNF-) having median values below the upper limit of normality. Thus, 26 (53%), 31 (63%), and 18 (37%) of patients had normal plasma IL-6, IL-1, and TNF- levels, respectively. Similarly, and despite the high dilution entailed in the lavage procedure, the levels of the different cytokines in BALF were significantly higher than in serum (Table 4). Individually, poor correlations were observed between serum and BALF levels, with a rho coefficient lower than 0.2 for all the cytokines evaluated except for IL-8 (r = 0.44, p < 0.002).

	DISCUSSION

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The present study has shown that in patients with clinically stable bronchiectasis: (1) there is an active neutrophilic inflammatory response in the airways that is present in patients with sterile bronchi but is exaggerated in those cases with MPP colonizing the airways; (2) colonizing bacteria seem to act as an inflammatory stimulus since the greater the bacterial load the greater the inflammatory response; (3) the bronchial inflammatory response seems to be compartmentalized and cannot be accurately evaluated in blood samples.

Airway Inflammation in Patients with Bronchiectasis

Patients with bronchiectasis in a stable clinical condition present a characteristic neutrophilic inflammation in the airways. These neutrophils are most probably activated since we also observed a significant increase in elastase and MPO concentrations in BALF. Other investigators have previously demonstrated a neutrophilic bronchial inflammation in patients with bronchiectasis. However, to our knowledge, this is the first report using BAL to evaluate inflammation (4, 15). Interestingly, our findings suggest that airway inflammation may occur even in the absence of colonization as demonstrated by the significant increase in levels of the different inflammatory mediators between patients with negative BAL cultures and control subjects. Although we cannot definitively exclude the theoretical presence of airway colonization in patients with negative BAL cultures, it is interesting to note that in a small series of infants with cystic fibrosis, Khan and colleagues (4) also found that the neutrophil count and the elastase/alpha₁-antiprotease complexes were elevated in patients with negative BALF cultures, reinforcing the concept that permanent airway inflammation not related to colonization may be present in the early stages of the disease.

It has been suggested that an imbalance between proinflammatory and antiinflammatory molecules determines the particular inflammatory response in the airways (8,16). Among the proinflammatory mediators involved, IL-8, IL-1, and TNF- most likely play a role favoring the trafficking of activated neutrophils through the bronchial wall into the bronchial lumen. More controversial is the role of the so-called anti-inflammatory mediators such as IL-6 and IL-10. These cytokines act as a counterpart of proinflammatory mediators by promoting the synthesis of natural antagonists of IL-1 and TNF- (17, 18). Interestingly, recent studies have shown low levels of IL-6 and IL-10 in the airway secretions of patients with cystic fibrosis, suggesting that a dysfunction in modulation of important homeostatic mechanisms may lead to the excessive inflammatory response observed in this disease (19). The increased concentrations of the chemoattractants IL-1, IL-8, and TNF- as well as the good correlations with the percentage of neutrophils recruited and their proteases observed in the present study, suggest that these mediators may have a preferential role in the inflammatory process. Interestingly, the levels of IL-6 and IL-10 were also increased compared with control subjects, although the differences did not reach statistical significance, probably because of the small number of control subjects evaluated.

Airway Inflammation and Colonization

Patients with MPP colonizing the airways had a more intense neutrophilic inflammatory reaction than did noncolonized patients (Table 3). It is of note that markers of inflammation increased progressively with the increase in the bacterial load reflected by the number of colony counts isolated by BAL (Figure 1). The relationship between bacterial load and the intensity of bronchial inflammation has already been observed in patients with chronic bronchitis and cystic fibrosis, confirming the potential role of bacteria in the genesis of inflammation and suggesting that antibiotic treatment directed towards the colonized flora may be potentially useful (20, 21). Although it has been speculated that microorganisms such as Pseudomonas spp. may cause a more intense bronchial inflammation (22), we did not see any differences in inflammatory parameters according to the type of bacteria isolated, though the number of patients with Pseudomonas spp. was too small to draw firm conclusions (data not shown). Although the population evaluated in the present study had fairly good lung function (mean FEV₁ of almost 80% predicted), cases with moderate airway obstruction had increased levels of different mediators in BALF, suggesting a more active inflammatory process in patients with more severe disease.

Systemic Evaluation of the Bronchial Inflammatory Process

The relatively low levels of plasma cytokines compared with BALF and the poor correlations between them suggest that the inflammatory process in the airways is mostly compartmentalized. Complex interactions between cytokines and their natural antagonists in the local milieu or the relatively low intensity of the bronchial inflammatory process may explain the apparent local effect observed in the present study. Interestingly, and according to our experience, the systemic reflection of local inflammatory response is closely related to the severity of the disease, being more intense in cases of severe pneumonia and ARDS (23, 24). Further studies are needed to confirm the compartmentalization of the inflammatory response in patients with bronchiectasis and to explore whether this response is isolated to the involved lung area or if it is generalized to the entire lung.

In summary, evidence that patients with bronchiectasis in a stable clinical condition present an active neutrophilic inflammatory reaction in the airways enhanced by the presence of MPP colonizing the airways and related to bacterial load suggest that therapeutic interventions aimed at treating the colonization and immunomodulatory therapies that can ameliorate the inflammatory process may hinder the progression of the disease.