All structures of the bronchi are involved in asthma: inflam- mation and remodeling processes occur in both large and small airways. Airway inflammation has ...
Assessment of Airway Inflammation in Asthma ANTONIO M. VIGNOLA, JEAN BOUSQUET, PASCAL CHANEZ, ROSALIA GAGLIARDO, ANNA M. MERENDINO, GIUSEPPINA CHIAPPARA, and GIOVANNI BONSIGNORE Istituto di Fisiopatologia Respiratoria, Palermo, Italy; and Service des Maladies Respiratoires, Centre Hospitalier Universitaire, CJF–INSERM U454, 34295-Montpellier-Cedex, France
All structures of the bronchi are involved in asthma: inflammation and remodeling processes occur in both large and small airways. Airway inflammation has been widely demonstrated in patients with chronic asthma, and it is correlated to the clinical severity of the disease (1, 2). However, inflammation is not restricted to the bronchi (3), and in the bronchial tree the exact location of airway inflammation is still a matter of controversy. It is, however, accepted that inflammation is exhibited in both large and small airways.
TECHNIQUES FOR ASSESSING AIRWAY INFLAMMATION The cellular, biochemical, immunologic, and molecular markers of airway inflammation can be studied by direct or indirect sampling procedures in the airways. Bronchoalveolar lavage, biopsies, bronchial brushing, and induced sputum are direct airway sampling procedures which have proved very useful for providing important information about the biologic mechanisms underlying asthma (4). However, inflammatory markers also can be measured in peripheral blood (5) or in urine (6). The noninvasive nature of these latter methods is a major advantage, but their indirect nature is a major limitation. There is no optimal assessment method; the choice depends upon the aims of the study. It seems clear that, in clinical practice, serial measurement of airway inflammation cannot be conducted with invasive methods. However, for research purposes, methods using fiberoptic bronchoscopy are appropriate. The role of sputum-based techniques is still under investigation. Whatever method is used, it is essential to evaluate the safety of the procedure, its reproducibility, repeatability, and sensitivity to change under the treatment studied. Sputum
Recently, the use of induced sputum has been proposed as a relatively standard method for research and assessment of antiasthma therapy (7, 8). In 1989, the Hamilton group proposed using hypertonic saline to improve sputum production when sputum could not be expectorated (7, 8). Once the sputum has been dissolved, cells can be enumerated using immunocytochemical methods or cytofluorimetry. Mediators such as eosinophil cationic protein (9, 10), histamine, tryptase, markers of mucus secretion (11), neutrophil-derived markers (12, 13) can be measured in the supernatants. The results of studies using induced sputum have provided evidence of reproducibility (14, 15), responsiveness (16, 17), and validation (14, 15). However, skilled investigators are required for this technique to provide satisfactory results.
Correspondence and requests for reprints should be addressed to A. M. Vignola, Istituto di Fisiopatologia Respiratoria, C.N.R., Via Trabucco 180, 90146 Palermo, Italy. Am J Respir Crit Care Med Vol 157. pp S184–S187, 1998
Bronchoalveolar Lavage
Bronchoalveolar lavage examines a poorly defined segment of the lung, including small and large airways in addition to alveoli (18). Because asthma is a bronchial disease, attempts have been made to obtain fluid derived from the bronchi only. In this regard, the use of a double-balloon bronchoscope or a double-balloon-tipped catheter inserted through a double-lumen bronchoscope (19, 20) seems to be the best method for performing a true bronchial lavage. Studies have shown that when the bronchial wash technique is used, there is a larger proportion of neutrophils and epithelial cells than in bronchoalveolar lavage. The protein profile differs when large and small airways are sampled separately (21). Although potentially useful for discriminating central from peripheral airway inflammation, bronchial wash techniques are more difficult to carry out than classical bronchoalveolar lavage because their safety has not been fully assessed and they cannot be performed on a large scale. Thus, other investigators (22–25) have proposed fractionating the lavage to obtain a first aliquot more related to a bronchial sample and other aliquots derived from more distal parts, including small airways and alveoli (the so-called “alveolar sample”). There are several arguments supporting the concept that the first aliquot of bronchoalveolar lavage is more related to a bronchial sampling: for example, the results have been found to be close to those obtained by the double-balloon technique (19, 20), and digital subtraction radiography has shown that although the first bronchoalveolar lavage aliquot remains close to the bronchoscope, the other aliquots spread more widely throughout the airways (26). These studies indicate that the bronchial sample should be analyzed separately from the subsequent aliquots more related to the distal parts of the airways and the lung. With regard to the repeatability of the method, it should be considered that allergen challenge may induce an inflammatory reaction for a period of a few weeks and, as in bronchial challenge performed using allergens, a second bronchoalveolar lavage cannot be carried out until 4 wk later. Nonspecific challenge with methacholine does not change the results of bronchoalveolar lavage fluid analyses (27), but histamine does (28). Bronchial Mucosal Biopsies
The first pathologic observations of patients who died from an asthma attack were made in the nineteenth century. However, until 1980 few pathologic studies were carried out in living patients with asthma. In the 1980s, fiber optic bronchoscopy made access to the bronchi easier, and techniques such as electron microscopy (27, 29, 30), immunohistochemistry (1, 31), double-staining immunohistochemical techniques (32), and in situ hybridization (33, 34) and other molecular biology–based techniques have significantly improved our knowledge (35). Bronchial biopsies offer several advantages (4, 36): (1) pathologic changes occurring in the mucosa of patients with asthma can be directly seen by sampling material from the local disease; (2) biopsy samples may be examined by all possible methods including electron microscopy, immunohistochem-
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istry, and molecular biology techniques; (3) after digestion, cell culture or measurement of inflammatory markers can be carried out; (4) the state of activation of cells and their secretory products can be observed; and (5) the localization of cells within the submucosa and epithelium can be analyzed. However, there are several disadvantages of bronchial biopsies. (1) Specimens may not be adequate to quantitate the inflammation of asthma, since they demonstrate only pathologic abnormalities of large airways (fourth to sixth generation bronchi). (2) Pediatric bronchoscopes may allow deeper sampling into the airways, but the size of biopsy is usually too small to make an analysis possible. (3) Also, because specimens are very small, they do not take into account the possible heterogeneity of the lesions. (4) Submucosal edema (due either to asthma or to the biopsic procedure) is often present, making quantitation difficult. (5) The specimens (particularly the epithelium) may be altered by the biopsic procedure. (6) Finally, at least 15 subjects need to be included in each group to obtain reproducible results. Transbronchial and Postmortem Biopsies
Postmortem (3) and transbronchial biopsy studies (37) suggest that the peribronchial tissue as well as the alveoli may be involved in lung inflammation in patients with asthma. A recent paper using transbronchial biopsies focused on the importance of lower airway inflammation (37), but two other studies on excised lung specimens showed that inflammation was similar in magnitude in the peripheral and central airways (38, 39). Bronchial Brushing
Bronchial brushing is another method for recovering bronchial epithelial cells (2, 40–43). Bronchial brushing makes it possible to obtain highly viable epithelial cells that can be analyzed using various cytologic methods including computerized interactive morphometry (44), immunocytochemistry (2), confocal microscopy (45), electron microscopy (46), flow cytometry (47), and DNA analysis (48). The metabolic activity of the cells obtained by brushing has been used to study their activation pattern in disease states (42) or their pharmacologic modulation (49). In patients with asthma, bronchial brushing has been used to study epithelial cells (2, 40, 42, 43) and inflammatory cells infiltrating the epithelium or present at the surface of the epithelial layer (50).
OVERVIEW OF CELLULAR INFLAMMATION Several cell types have been characterized in the inflammatory infiltrate of the asthmatic airway (51). These include inflammatory cells such as eosinophils, mast cells, macrophages, T cells, and neutrophils (52); structural cells such as fibroblasts and smooth muscle cells; and epithelial cells, which are of particular importance in the interactions between the ambient air and the submucosa. These cells participate in the release of vasoactive mediators, toxic metabolites, and cytokines that are involved in acute and chronic bronchoconstriction. Several cell types can release regulatory cytokines, modulating and perpetuating the inflammation in the airways. More recently, attention has been focused toward airway remodeling in which growth factors, extracellular matrix components, and metalloproteases and their inhibitors (TIMPs) play a complex role (53).
of goblet cells. It has a fragile appearance; the ciliated cells look swollen, vacuolized, and often show loss of cilia (29). Epithelial cells are significantly less viable in patients with asthma than in subjects without it (42). These cells appear activated as they release a greater amount of hydroxyeicosatetraenoic acid (15-HETE), prostaglandin E2, fibronectin, and endothelin, either spontaneously or after stimulation (42, 56), and they present an increased expression of membrane markers (2), cytokines (57) or chemokines (58). In asthma, epithelial cells can be activated by immunoglobulin E–dependent mechanisms (59) or pro-inflammatory mediators such as histamine (49). Activated epithelial cells release a wide array of mediators including 15-HETE (42, 60), cytokines (61), or extracellular matrix proteins (42) that can induce bronchial obstruction, inflammation, and airway remodeling. Eosinophils. Activated eosinophils are found in the airways of people with symptomatic asthma (62). In chronic asthma, eosinophils have been found in increased numbers in bronchial biopsies (1). These cells are usually in an activated stage and located beneath the basement membrane. Most allergic and nonalleric patients with asthma, including those with mild asthma, have eosinophils in their bronchi; there is a significant correlation between the activation of eosinophils and the severity of asthma (1). However, bronchial eosinophilia is not a unique characteristic of asthma because eosinophils can be observed in many diseases, including chronic bronchitis (63). Lymphocytes. T lymphocytes are another major cell type of the mixed cell infiltrate present in the airways of patients who have died from an asthma exacerbation (64) or patients of variable etiology, including occupational asthma (65, 66). They often bear CD4 receptors, whereas CD8-positive cells are more rarely identified, even during exacerbations (67). There is a correlation between the severity of asthma and the number of CD4-positive cells in biopsies. T cells are likely to play an important role in asthma. Using in situ hybridization techniques, a Th2 phenotype of T cells has been observed in the bronchoalveolar lavage fluid of asthmatics. After allergen challenge, many allergen-specific T cells bear the Th2 phenotype in bronchial biopsies or bronchoalveolar lavage fluid (66, 68). Mast cells. Metachromatic cells are found in the bronchi of subjects with or without asthma (69, 70). However, they are degranulated in the airways of patients with asthma as shown directly by electron microscopy (29) and immunocytochemistry and indirectly by increased levels of tryptase, histamine, and prostaglandin D2 in the bronchoalveolar lavage fluid (71). Moreover, a significant correlation with the severity of asthma and the level of histamine or tryptase in the bronchoalveolar lavage fluid has been reported (71). Macrophages
Mononuclear phagocytes are likely to be involved in the pathogenesis of asthma (72), since macrophages are among the cells present in the airway’s inflammatory infiltrate (73). Alveolar macrophages recovered by bronchoalveolar lavage have been extensively studied in asthma. Most studies have revealed increased alveolar macrophage activation (74, 75), which was found to be significantly correlated with the severity of asthma in some studies (76). Using Percoll density fractionation, it was shown that alveolar macrophages of patients with asthma are hypodense compared with those of normal subjects (77).
Epithelium
CONCLUSION
Epithelium is shed (54) and activated but its regeneration appears to be normal (55). When epithelial cells are present, the epithelium is still pseudostratified with an increased number
All cells of the airways can actively participate in the pathogenesis of bronchial inflammation in asthma, a disease that appears to be far more complex than a simple eosinophilic disorder.
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