Eur Resplr J, 1993, 6, 461-464 Printed In UK • all rights reserved

EDITORIAL

Copyright ~ERS Journals Ltd 1993 European Respiratory Journal

ISSN 0903 • 1936

Assessment of airways Inflammation in chronic bronchitis

AB. Thompson, S.l. Rennard*

Chronic bronchitis is a clinical syndrome, defined on the basis of sputum production [1, 2]. Investigations have demonstrated that airway inflammation is central to the clinical changes associated with chronic bronchitis. His­tological investigations of surgical and autopsy specimens have provided material to determine the site and nature of the airway inflammation. Sampling of the airways via flex ible fibreoptic bronchoscopy has also advanced under­standing of the nosology and cell biology of airway inflammation, and promises to provide a means for ra­tional assessment of therapeutic interventions.

Histological investigations of resected lungs, and lungs obtained at autopsy, have correlated the presence of air­ways inflammation with airways obstruction in chronic bronchitis. In studies reported by Costo et al. [3], air­way inflammation was found to be a distinguishing char­acteristic in the airways of smokers with obstruction, compared to smokers without obstruction. More recently, these findings have been confirmed by MUUEN et al. (4], who demonstrated that airway Infl ammation correlated with hypertrophy of mucous glands and best distinguished smokers with chronic bronchitis from smokers without chronic bronchitis. Thus, pathological investigations con­firm the central role of airways inflammation in the clini­cal manifestations of chronic obstructive. pulmonary disease (COPD). However, pathological investigations are obviously limited in their applicability for testing of thera­peutic modalities directed towards ·the treatment of airways inflammation.

Sputum has been used for assessment of airways in­flammation in chronic bronchitis [5]. In particular, spu­tum cytology has been used to determine the presence or absence of intraluminal neutrophilia, and can, to some degree, assess the intensity of the inflammation. Moreo· ver, mediators of airways inflammation can be assessed in sputum [6, 7]. However, measurements in sputum are limited by several factors. Sputum is not available from normal individuals. The collection of expectorated spu· tum is inevitably associated with contamination by oral contents. Sputum is very heterogeneous. Finally, quanti-

• S.I. Rennard, Section of Pulmonary and Critical Care Medicine, Dept of Internal Medicine, University of Nebraska Medical Center, 600 S 42nd St., Omaha, NE 68198-5300, USA.

fication of sputum contents is difficult; sputum is diffi­cult to physically manipulate, and measurements ex­pressed as concentrations may not in fact accurately reflect the degree of hydration of the sputum, the extent of contamination with saliva, or the rate of clearance of sputum from the lower respiratory tract. In spite of these limitations, sputum has been successfully employed to assess airways inflammation, and to measure the effect of therapeutic interventions upon lower respiratory tract inflammation [8, 9].

Alternatively, bronchoscopic techniques can be used to sample the lower respiratory tract. Flexible fibreoptic bronchoscopy allows direct visualization of the mucosa of the first four to six generations of the airways. Con­sistent with inflammation elsewhere, inflammation of mucosal surfaces is associated with erythema and oedema. In addition, when inflamed, mucosal surfaces increase secretion and become more susceptible to physical trauma [10] . Taking advantage of these characteristics, a semi­quantitative scale has been developed for the visual as­sessment of airways inflammation [11]. Using the "bronchitis index", cigarette smokers who deny symptoms of chronic bronchitis have been found to have abnormal appearance of the airways (mean=8.5, range=4-18) com­pared to normals (mean=2.3, range=0-12), but have sig­nificantly less severely abnormal appearance of the airways compared to smokers with chronic bronchitis (mean=12.2, range=3-31) [12]. In addition, the bronchi­tis index has been noted to improve with therapeutic interventions [13, 14).

Bronchoalveolar lavage (BAL) has been used to quantitate the cellular and biochemical markers of airways inflammation in a number of airway diseases, including chronic bronchitis, asthma, cystic fibrosis and bronchiecta­sis (in normal hosts). Bronchoalveolar lavage can be per­formed in a manner which allows for enrichment with airways contents. It has been demonstrated that the ini­tial fluid infused, following wedging of the bronchoscope into an airway, tends to preferentially sample the airways [15, 16]. This preferential sampling is dependent upon in­fusion of small volumes. The adequacy of the preferen­tial sampling of airways is reflected by BAL fluid content with greater numbers of airway epithelial cells [17], and higher concentrations of proteins such as lactoferrin and lysozyme, which are secreted by airway serous cells, than

462 A.B. THOMPSON, S.I. RENNARD

in the alveolar sample (18]. Using such criteria, volumes of 20 ml or less provide samples enriched, perhaps ten­fold, with airway contents.

Cigarette smoking and chronic bronchitis have both been shown to influence BAL findings (19]. Broncho­alveolar lavage in young smokers has demonstrated elevated recovery of lavage fluid cells, in particular macrophages and neutrophils [20]. MARTIN et al. [21] demonstrated that chronic bronchitis was associated with neutrophilic inflammation, and that the first instilled 50 ml of lavage fluid contained higher amounts of neutrophils than later instilled aliquots. Employing 20 ml aliquots to sample airways contents has demonstrated that chronic bronchitis is associated with airways neutrophilia (12). The intensity of the airway neutrophilia was posi­tively correlated with airways obstruction, sputum produc­tion, and smoking history. In a separate study, an elevated proportion of goblet cells, presumably reflecting goblet cell metaplasia, was recovered by lavage of chronic bronchitics (22]. The number of goblet cells was strongly correlated with the forced expiratory volume in one sec­ond (FEV1), corroborating previous observations (23]. Furthermore, BAL has disclosed elevated airway albumin [12], and proteins felt to be derived from airways epi­thelial serous cells, lactoferrin and lysozyme [18], in association with chronic bronchitis. Therapeutic interven­tions, including smoking reduction [13), theophylline [24), and inhaled beclomethasone (14), have been shown to ameliorate these indices of airway inflammation in chronic bronchitis.

Bronchoalveolar lavage is not without its limitations. It is clearly more invasive than collection of expectorated sputum. No standard technique is universally accepted for the performance of BAL [25, 26). A number of differ­ent techniques have been employed by investigators, which may account for slight differences in results. In­deed, differences resulting from technical considerations have been advantageously exploited by investigators. A more important limitation is that there is no gold stand­ard for the reporting of BAL data. The variable dilution of the epithelial lining fluid of the lower respiratory tract during BAL remains problematical. Various endogenous markers for dilution have been used, particularity albu­min and urea [27]. However, both the concentration of albumin [14, 28], and of urea in BAL fluid are subject to systematic errors (29, 30). One approach to this di­lemma is to express data as a concentration of lavage fluid, normalized to albumin, and as an estimated con­centration in epithelial lining fluid, using the urea method (31]. Expressed in this manner, and taking into account the limitations of albumin and urea as endogenous mark­ers for dilution, comparisons can be made between sub­ject groups.

Sampling of the endobronchial mucosa can be per­formed via the flexible fibreoptic bronchoscope, by di­rect brushing or biopsy. Brushing the bronchial wall with a cytological brush, and agitating the cells in saline or medium, yields dispersed cells and clumps of bronchial mucosal cells. The cells can be further dispersed with proteases, or manipulated as clumps. The epithelial cells can be quantitated and freshly preserved, or maintained

in cultures. Advances in molecular biology and immu­nohistochemistry have produced very sensitive techniques, which can be fruitfully applied to the epithelial cells har­vested by endobronchial brushing.

Endobronchial brushing is slightly more invasive than BAL, and is limited by the relatively small area of en­dobronchial anatomy which can be sampled. However, brushing provides a representative cross-section of epithe­lial cells, directly sampled in situ. Thus, investigations of cells sampled by brushing do not suffer the experimen­tal artifacts theoretically present for BAL, which harvests a population of epithelial cells that are presumably loosely adherent or non-viable. Endobronchial brushing of sub­jects with chronic bronchitis has disclosed an increased percentage of epithelial goblet cells, compared to normal nonsmokers [32]. The sensitive methodologies of molecu­lar biology are ideally suited for the investigation of cells obtained by brushing. Ribonucleic acid (RNA) has been extracted from the cells and probed with reagents to de­tect messenger ribonucleic acid (mRNA) for inflamma­tory mediators [33], and brushed epithelial cells have been used as targets for transfection experiments [34]. Immu­nohistochemistry can be applied to delineate both mem­brane-bound and cytoplasmic antigens.

Sampling of the airway epithelium by endobronchial biopsy adds a further degree of invasiveness, and is even more limited in the surface area of bronchial mucosa which is sampled. However, endobronchial biopsy has the unique property of preserving mucosal anatomy. This property of biopsies permits assessment of pathological changes, which are not appreciated by brushings. Biop­sies can also be used to quantitate inflammatory cells within the mucosa and adjacent submucosa. As for the epithelial cells obtained by brushing, endobronchial biop­sies provide effective samples for immunohistochemistry and molecular biology.

Bronchial biopsy has been used to good advantage, especially for the investigation of airway inflammation as­sociated with asthma. Biopsies have confirmed the involvement of eosinophils, even in mild asthmatics [35]. In mild asthma endobronchial biopsies have indicated ul­trastructural changes suggestive of epithelial fragility with hyperreactivity [36]. Endobronchial biopsy has also proved to be of value in the assessment of therapeutic interventions. Biopsies performed before and after 10 yrs of daily therapy with inhaled steroids demonstrate reso­lution of inflammation and reduced epithelial damage (37]. The successful application of endobronchial biop­sies in the study of asthma holds promise for the wider application of invasive sampling of bronchial epithelium for therapeutic investigations in other airway inflamma­tory diseases, such as chronic bronchitis and cystic fibro­sis.

As newer therapeutic strategies for the treatment of lower respiratory tract inflammation are developed, the importance of the assessment of airways inflammation and its response to therapy will continue to grow. In sub­jects with chronic bronchitis and fixed obstruction, demonstrations of therapeutic efficacy by means of tra­ditional pulmonary function tests would require long-term investigations with large numbers of subjects. In

ASSESSMENT OF AIRWAYS INFLAMMATION 463

contrast, the demonstration of amelioration of airways in­flammation has been shown to be a practical alternative. Improvement in morbidity and mortality are, of course, the ultimate yardsticks for therapeutic efficacy in COPD. Demonstration of efficacy in the therapy of airways in­flammation may, however, provide the rationale and jus­tification for extended longitudinal studies for specific therapeutic interventions.

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