Investigative Use of Bronchoscopy in Asthma

NIZAR N. JARJOUR, STEPHEN P. PETERS, RATKO DJUKANOVIC>, and WILLIAM J. CALHOUN

From the Section of Pulmonary and Critical Care Medicine, Department of Medicine, University of Wisconsin School of Medicine, Madison, Wisconsin; the Section of Pulmonary Medicine and Critical Care, Jefferson Medical College, Philadelphia, Pennsylvania; the School of Medicine, University Medicine, Southampton University General Hospital, Southampton, United Kingdom; and the Division of Pulmonary, Allergy and Critical Care Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania

	INTRODUCTION

TOP INTRODUCTION CONCLUSION REFERENCES

In the early 1970s, Reynolds and Newball (1) were among the first to report their experience with fiberoptic bronchoscopy and "bronchial" (bronchoalveolar) lavage (BAL) in order to obtain respiratory cells and secretions from human volunteers for in vitro analysis. However, it was not until approximately 10 years later that studies were initiated in subjects with asthma (2). Since then hundreds of studies have been done using bronchoscopy and various bronchoscopic techniques (reviewed in part in reference 3). These investigations demonstrated the importance of airway inflammation in asthma (4), even in patients with mild disease, and supported the recommended use of antiinflammatory medications in asthma (5). In this review, we summarize information concerning the investigative use of bronchoscopy in asthma, with special emphasis on the methods, safety, and utility of BAL, antigen challenge, endobronchial biopsies, and bronchial brushing.

	PATIENT ASSESSMENT AND PREPARATION FOR BRONCHOSCOPY

Bronchoscopy can be associated with serious complications, of which bronchial obstruction is of particular relevance to asthma. Published guidelines recommend that a thorough evaluation of each prospective subject, including a medical history, focused physical examination, and spirometry, be obtained by the investigator prior to bronchoscopy (6). Coagulation studies (prothrombin time [PT], partial thromboplastin time [PTT], and platelet count) are generally performed prior to biopsy (especially transbronchial) procedures. A route for injection of intravenous medications is recommended (7), especially if intravenous sedation is to be used. However, when no sedation is used or when minimal sedation is given through intramuscular route, an intravenous catheter might not be necessary in most subjects. In severe asthma this precaution might still be warranted. Premedication with atropine, bronchodilators, and sedatives can be either administered or omitted, depending on the specific research protocol and subject comfort and safety (7). Finally, minimal doses of topical anesthetic should be used to achieve patient comfort and cough control. Table 1 summarizes the important aspects of evaluating and monitoring subjects undergoing investigative bronchoscopy.

	BRONCHOALVEOLAR LAVAGE

Utility

Studies of BAL in asthma have revealed increased numbers and activation of inflammatory cells and higher levels of mediators when compared to subjects without asthma. Asthma subjects show increased numbers of BAL eosinophils, even when the disease is mild and stable (8, 9). Increased asthma severity is associated with increased numbers and activation of airway eosinophils (8). However, correlations between numbers of eosinophils and airway obstruction and hyperreactivity are often difficult to demonstrate. Macrophage activation has been described in symptomatic asthma and with nocturnal exacerbations (10, 11). While BAL neutrophilia is not a predominant feature of mild to moderate asthma, it has been described in response to occupational challenge with isocyanate or grain dust (12) and endotoxin-contaminated allergen extracts (13). There is some evidence to suggest that patients with severe asthma might have neutrophilic airway inflammation (14). Many mediators and cytokines have been detected in BAL fluid of asthma patients, including interleukin (IL)-1, IL-2, IL-4, IL-5, IL-6, IL-10, granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor (TNF-) (15). Other studies have noted increased levels of mRNA for several cytokines in BAL cells, including TNF-, GM-CSF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-5, IL-6, and IL-13 (15). Interleukin-5 and GM-CSF increased significantly following airway allergen challenge, in correlation with the influx of eosinophils (16, 17). Other mediators detected in BAL fluid include leukotrienes, prostaglandins, histamine, tryptase, and soluble adhesion molecules. Bronchoalveolar lavage has also been performed to evaluate the effects of asthma therapies (corticosteroids, theophylline, beta-agonists, cromolyn sodium, nedocromil sodium, cetirizine, leukotriene inhibitors, etc.) on parameters of airway inflammation. These studies have enhanced our understanding of the mechanisms of asthma and the specific role of some of the mediators (e.g., leukotrienes) in asthma.

Techniques

The details of the techniques used to perform BAL vary widely among research groups. Generally, the fiberoptic bronchoscope is introduced after light premedication and topical anesthesia of the nose and upper airway. The tip of the bronchoscope is wedged into a segmental, or subsegmental, bronchus. The quantity of fluid used in lavage varies from 100-400 ml of normal saline. The fluid should be sterile and injected in aliquots of 20-60 ml. The saline should be warmed to body temperature to avoid thermally induced bronchoconstriction in asthma subjects. Up to four separate segments have been lavaged (lavage volume of 120 ml in each) in one setting without adverse events (16, 17). Gentle intermittent suction should be used to recover the effluent. Although periodic deep breathing has been recommended to enhance fluid recovery, this can lead to fluid leak, which may trigger cough. Fluid recovery of 60-80% is expected in subjects with mild asthma; however, the recovery is around 50% in those with moderate to severe disease. Some investigators separate the return from the first aliquot as "bronchial" lavage, and pool the return from the remaining aliquots as "alveolar" sample. Bronchoalveolar lavage cells are typically removed by centrifugation, enumerated, and a differential count is prepared. Proteins, mediators, immune globulins, cytokines and many other substances have been measured in BAL fluid. The results are most often reported "per ml of BAL fluid"; however, there is no universally acceptable method to report the results and correct for the degree of epithelial lining fluid dilution.

Safety

The first National Institutes of Health (NIH) workshop on the investigative use of bronchoscopy in asthma concluded that BAL can be performed in mild asthma with minimal risk to subjects (6). By 1990 more than 200 papers had been published on bronchoscopy in asthma. The accumulated safety data allowed for the revision of the prior guidelines to include subjects with more severe disease (FEV₁ < 60%), repeated lavages, endobronchial biopsies, and local allergen challenge (7). Since then, several studies have confirmed the safety of BAL in asthma (18). Bronchoscopy with BAL can be associated with hypoxemia, cough, wheezing, and post-bronchoscopy fever. Some investigators found hypoxemia to be more severe in asthmatics compared to normal subjects (18), while others did not (19). Furthermore, hypoxemia is rarely severe enough to require premature termination of the procedure and can usually be treated or prevented by supplemental oxygen (19). Cough during the procedure is common and often responds to topical anesthesia. Wheezing can occur during bronchoscopy, especially in patients with more severe asthma, when local antigen challenge is done (21) or when premedication with beta-agonist is omitted (22). While patients with low FEV₁ and/or severe airway responsiveness might be at increased risk for developing bronchospasm (18), BAL does not lead to sustained exacerbation of airway obstruction (22), worsening of bronchial hyperresponsiveness (20), or diffuse airway inflammation (22). Post-bronchoscopy fever (high temperature [38.5-39.5° C], chest pain, body aches) is rarely seen after BAL. When it occurs, it starts several hours following the procedure and usually resolves within 24-48 h. Sedation and topical anesthetics used with bronchoscopy can also have side effects and complications, such as respiratory depression and lidocaine overdose. Circulating levels of lidocaine are probably influenced by site of delivery (mouth, tracheobronchial tree), presence of airway inflammation (potentially increased absorption), duration of the procedure, and the total lidocaine dose. A maximum dose of 400 mg was recommended in the first NIH guidelines (6); however, the revised guidelines did not specify an upper limit but recommended minimizing the total amount used (7).

Conclusion

In most patients, bronchoscopy with BAL can be performed safely, provided appropriate guidelines are followed. The procedure should be undertaken only when a clear hypothesis is being tested and the necessary technical expertise, monitoring equipment, and the ability to treat potential complications are immediately available. Patients should receive premedications with sedative (e.g., midazolam, fentanyl), atropine (or glycopyrolate) and beta-agonist (inhaled or nebulized). Some or all of these premedications can be omitted if necessary for specific studies, such as those evaluating the immediate response to antigen. The lavage should be limited to four segments or less and the total lavage volume to 480 ml or less. Patients should be monitored clinically and by pulse oximetry during the procedure and for at least 1 h following the procedure. Investigators need to minimize the total amount of sedatives and lidocaine used while still maintaining comfort and safety.

	SEGMENTAL ALLERGEN CHALLENGE

Utility

Airway inflammation plays a major role in the pathogenesis of asthma. The majority of patients with asthma have allergic sensitivities that can be used to provoke airway inflammation. Aerosolized allergen challenge results in the development of bronchial obstruction, airway hyperresponsiveness, eosinophilic infiltration and activation, increased superoxide production, and other indices of inflammation (23). However, aerosol challenge has both conceptual and practical problems. First, only a small and variable fraction of the dose delivered by the nebulizer actually reaches the lower airway. Moreover, the site of deposition is unknown unless radiotracers are used, and in any case is uncontrolled. Finally, the quantity of allergen that can be delivered is limited by safety considerations due to airway obstruction.

Segmental allergen challenge (SAC) addresses many of these limitations. Allergen dosing and localization are more precise, because allergen is delivered to a specific airway segment under direct visualization. Multiple segments can safely be challenged with different antigen doses (for dose-response studies), or the same dose can be used in multiple segments (for kinetic or interventional studies) (26). The degree of generalized bronchial obstruction is less than that seen with aerosol challenge, and the degree of inflammation is greater (29). The site of the inflammatory response is reasonably localized, particularly if careful attention to technique is given. Instrumentation and BAL per se do not evoke generalized pulmonary inflammation (10, 22). Finally, SAC localizes allergen challenge to the small airways, which are sites of inflammation (30, 31) and bronchial obstruction in asthma (32). While SAC is a powerful tool for studying allergen-driven airway inflammation, care should be taken when extrapolating SAC findings to natural exacerbations of asthma.

This technique has provided unique insights into the differences between asthmatic and nonasthmatic atopic subjects. Wenzel and colleagues (33) showed that the concentration of leukotriene C₄ was significantly greater after endobronchial allergen challenge in asthmatics compared to nonasthmatic atopics. Preliminary data suggest that failure to secrete IL-10 after allergen challenge might also differentiate allergic asthma from allergic rhinitis subjects (34). Recently, Shaver and colleagues (35) reported that the increase in BAL eosinophils and IL-5 in response to antigen SAC were similar in allergic asthmatic and allergic nonasthmatic subjects. Interestingly, subjects with documented late asthmatic response to whole lung challenge showed the strongest cellular and cytokine responses to SAC (35).

Techniques

Several methods have been used to select antigen dose for use in SAC. These include a uniform dosing (36), titrating antigen dose to cause a targeted degree of airway obstruction (37), various skin test titration methods (38) and inhaled allergen challenge (16, 17, 26, 29, 41, 42). Antigen dose is delivered in volumes of 5-20 ml (17, 27, 33, 39), while the bronchoscope is being held in wedge position to minimize spillage. Bronchoalveolar lavage is performed minutes, (16, 17, 33, 38, 41) 3-24 h, (28, 43) or 2-16 d (28, 37) following SAC. BAL analysis has confirmed the release of mast cell mediators immediately, neutrophilia in the first few hours, and eosinophilia 1-16 d after SAC.

Safety

Segmental allergen challenge is well tolerated. Cough and mild bronchospasm have been reported (38). The reduction in FEV₁ within 2 h following SAC ranged from 10-35% (16, 21, 37, 43). This obstruction persists in some studies but at smaller magnitude 48 h following SAC (23). Hypoxemia can occur but is usually mild or responds well to supplemental oxygen.

Conclusion

Segmental allergen challenge resulted in enhanced understanding of the pathogenesis of allergen-driven airway inflammation. Precautions for ensuring the safety of volunteers are similar to those for bronchoscopy and BAL alone. The choice of specific allergen should be based on history and skin testing. Different methods for dose selection have been used (see above). Timing of recovery of initial samples is probably immaterial so long as sufficient time has elapsed for allergen binding and mast cell activation (3-5 min). Patient comfort and logistics will likely limit analysis of the early response to within 15-20 min of SAC. More varied will be the timing of BAL to assess late responses, which will depend on the specific hypotheses being tested.

	BRONCHIAL BIOPSY

Utility

The use of bronchial biopsy for research purposes in asthma was first reported in 1977 (44). Initially believed to be a somewhat aggressive method, bronchial biopsy is now a routine investigative tool that is widely used in asthma research. Bronchial biopsy provides valuable insight into the morphology of the asthmatic airways, enabling detailed study of the epithelium, basement membrane, and submucosa (45). This has resulted in invaluable information about increased deposition of collagen within the reticular layer of the basement membrane. Furthermore, immunohistochemical studies of bronchial biopsies have enabled quantification of the inflammatory cells, such as mast cells, eosinophils, and T lymphocytes, and the extent of activation of these cells (46, 47). More recently, the production of cytokines has been studied using a combination of immunohistochemistry to identify the protein and in situ hybridization to look for evidence of activation of the genes for these cytokines (48). Together these studies have defined asthma as an eosinophilic bronchitis that is driven by an excessive production of Th2-type cytokines, and are now focusing attention on determinants of disease severity and airway remodeling. Using transmission electron microscopy, it has been possible to study the ultrastructure of inflammatory cells (47), and recently the immunogold technique has localized cytokines to ultrastructural elements of cells and adhesion molecules within the epithelium (49). Furthermore, the study of mucosal nerves and their neuropeptide products has improved our understanding of the neural mechanisms that contribute to asthma pathogenesis (50). Finally, Kraft and colleagues (31) recently reported the first study in which transbronchial biopsies were obtained in asthma subjects. Prominent tissue eosinophilia was seen at 4:00 A.M. in asthma subjects with nocturnal decline in pulmonary functions compared to asthmatic subjects without nocturnal asthma.

Techniques

The guidelines on the investigative use of bronchoscopy in asthma research have so far recommended that a maximum of six bronchial biopsies be taken (7). If performed in association with BAL, bronchial biopsies are usually taken following this procedure since a small amount of blood can contaminate the BAL fluid. Biopsies can be taken with cupped, fenestrated forceps, although several authors prefer the use of alligator forceps (e.g., Olympus FB15C). Samples are taken at bifurcations of segmental and subsegmental airways, although some studies have also taken specimens from the main carina. Biopsies should be processed immediately by either snap-freezing or embedding into paraffin or resins such as araldite and glycol methacrylate. The choice will depend on which methods of analysis are to be employed. Because paraffin and resin sections are thinner than frozen ones, they give better resolution and morphology, but frozen sections are probably best suited for in situ hybridization. The use of glycol methacrylate enables adjacent thin (2 µm) sections to be used to co-localize immunostaining with different antibodies to the same cell. This is not possible with frozen biopsies because no more than one section can be cut through a cell layer. Frozen biopsies can also be used to analyze mRNA expression for cytokines/ chemokines or adhesion molecules using the method of reverse transcription polymerase chain reaction (RT-PCR).

Safety

Since the introduction of bronchial biopsy via the fiberoptic bronchoscope into asthma research, there have been no reports of associated serious adverse events. The safety of bronchial biopsies in subjects with asthma has been the focus of several reports. The first study found that the combination of BAL and biopsy induced significant falls in FEV₁ in both normal and asthmatic subjects, although the reduction was more pronounced in those with asthma (18). A more recent study of subjects with mild asthma (51) found that bronchial biopsy does not affect disease activity, as measured by daily peak expiratory flow (PEF), usage of ₂-agonists, or asthma symptoms recorded over 14 d. This study also found that the degree of arterial O₂ desaturation and the extent of reduction in peak flow were similar in asthmatic and normal subjects. The authors also found a trend toward a smaller reduction in peak expiratory flow rate when only biopsies were taken as compared with the combination of biopsy and BAL. At least two studies (18, 51) have found a relationship between airway hyperresponsiveness (as measured by PC₂₀) recorded before bronchoscopy and the maximum fall in PEF after bronchoscopy, and in one of the studies this association was seen only when biopsy was performed in association with BAL and not when it was conducted in isolation (51). Bronchial biopsy is well tolerated even in subjects who do not receive premedication with bronchodilators (19); however, omitting bronchodilators can be associated with a more severe airway obstruction after bronchoscopy, which requires added caution. To date, transbronchial biopsy in an asthmatic subject has been done only by one group of investigators (31). In that study one subject (out of 21) developed a 10% pneumothorax, which rapidly resolved with conservative therapy (31). Therefore, the procedure seems to be well tolerated in asthmatic subjects.

Conclusion

Bronchial biopsies should be conducted by experienced bronchoscopists. The threshold for aborting the procedure should be low to ensure subject safety. Although a maximum of six biopsies were recommended in 1997 (7), experience since then suggests that up to 10 bronchial biopsies can be obtained safety, preferably from the same lung (51). Distal biopsies should be taken with small forceps (either cupped, fenestrated, or alligator) and larger subcarinae can be sampled with a relatively large forceps (e.g., Olympus FB32C or FB37K). If taken in association with BAL, biopsies should be performed following BAL. Because of the limited data, added caution is recommended when contemplating studies that require transbronchial biopsies.

	BRONCHIAL BRUSHING

Utility

While the technique of endobronchial biopsy has provided much of the key information to define airway inflammation characteristic of asthma, the techniques of BAL and bronchial brushing not only provide complementary histologic information but also cells for in vitro study. The majority of cells obtained by brushing are bronchial epithelial cells, which are now recognized to be more than just a lining of the airway mucosa. Bronchial epithelial cells release inflammatory mediators and participate in airway immune responses as antigen- presenting cells. Bronchial epithelial cells from asthmatic subjects have also been shown to have enhanced expression of HLA-DR and ICAM-1 (i.e., more activated) compared with normal subjects (52). They can also release mediators relevant to the pathogenesis of asthma, such as 15-hydroxy-eicosatetranoic acid (15 HETE), prostaglandin E₂, and fibronectin (53). Epithelial cells from asthmatic subjects have also enhanced expression of GM-CSF, which decreases following treatment with inhaled steroids (54).

Methods

Investigators from various laboratories have used different techniques for harvesting, preparing, and culturing human bronchial epithelial cells obtained by brushing. For example, Kelsen and coworkers (55, 56) harvested cells from the lower trachea of normal subjects while avoiding lidocaine anesthesia below the vocal cords in order to improve the viability of recovered cells. Using this technique, they reported a yield of approximately 3 million cells with 93% viability (56). Hastie and colleagues (57) and Penn and coworkers (58) have performed brushings of subsegmental airways after lavaging the same segment. The latter procedure has the advantages of removing lidocaine, which can be toxic to epithelial cells (55), and removing contaminating inflammatory cells, a consideration of major importance when epithelial cells are obtained from a lung segment that has undergone prior SAC (57, 58). Vignola and colleagues (52) performed brushing from two different subsegmental bronchi in the left lower lobe; in each place 10 gentle upward and downward strokes were done.

Several methods for manipulation of human bronchial epithelial cells have been used. One results in a preparation of single cells that is suitable for receptor binding studies and employs a brief trypsinization step (0.05% trypsin, 5 mM EDTA, 37° C, 2 min [58]). Another method, which avoids enzyme treatment and therefore "preserves" some of the cell clumps that are present in the initial cell preparation after cells are removed from the brush, is useful to analyze epithelial cell ciliary beat frequency and for short-term cell culture (57). Culturing the cells in Millicell-CM culture inserts (0.4 µM pore size; Millepore Co., Bedford, MA) with appropriate medium added to the outer well allows for studies of the effect of other cells, such as inflammatory cells obtained by BAL, on epithelial cell expression of genes and proteins without contaminating the epithelial cells. Additional manipulations can be done as experimental protocols demand. A third method involves collecting epithelial cells in tubes containing RPMI and antibiotics. The cells are then pelleted by centrifugation, washed, and resuspended in RPMI (52). The cell suspension can then be used for preparing cytospins or flow cytometric analysis.

Safety

Bronchial brushing is generally safe (52, 53). Bleeding is a potential, but rare, complication. Cough is another potential adverse effect; however, it is typically short-lived and resolves once brushing is completed.

Conclusion

Bronchial brushing has contributed significantly to our understanding of the role of epithelial cells in airway disease. Bronchial brushing has been obtained from subsegmental bronchi (53, 54, 58, 60). Four to 10 long, gentle strokes of a brush, two to four brushes per segment, and one to two segments per bronchoscopic procedure have been safely performed. The 1991 NIH guidelines recommended limiting bronchial brushing to two to four areas (7). Cell recovery is around 3 million, and the viability is typically 50 to 80%, which is increased by procedures used to prepare single cell suspensions (58); viable cells typically remain viable after overnight culture (57).

	SUMMARY

TOP INTRODUCTION CONCLUSION REFERENCES

The incorporation of bronchoscopy and bronchoscopic procedures into the investigation of asthma mechanisms, treatment, and in particular, the role of airway inflammation has contributed significantly to the enhanced understanding of this disease. Carefully drafted guidelines have allowed the gradual inclusion of subjects with more severe disease in studies utilizing bronchoscopic investigative tools. Many more questions remained unanswered, including the importance of persistence of airway inflammation in asymptomatic asthma, the specific antiinflammatory effects of new (and old) asthma therapies, the contribution of airway structural changes (subepithelial fibrosis) to nonreversible obstruction, the role of antiinflammatory versus proinflammatory cytokines in the pathogenesis of airway inflammation and the heterogeneity of disease expression in various groups of subjects. We are confident that current and future meticulously designed and executed research studies utilizing bronchoscopic techniques will significantly add to our knowledge of disease mechanisms and lead us to new and improved treatments for asthma.

	Footnotes

Correspondence and requests for reprints should be addressed to Nizar N. Jarjour, M.D., University of Wisconsin Hospital and Clinics, 600 Highland Ave., Madison, WI 53792.

(Received in original form May 8, 1997 and in revised form September 24, 1997).

Acknowledgments: Supported by NIH Grants AI24509, HL-2803, and HL42242.

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