ROLE OF BRONCHOSCOPY IN ASTHMA RESEARCH

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FLEXIBLE BRONCHOSCOPY IN THE 21ST CENTURY

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ROLE OF BRONCHOSCOPY IN ASTHMA RESEARCH Mani S. Kavuru, MD, Raed A. Dweik, MD, and Mary Jane Thomassen, PhD

Flexible bronchoscopy (FB) and associated techniques [bronchoalveolar lavage (BAL), mucosal brushing, endobronchial biopsy, airway nitric oxide measurement] have contributed substantially to our understanding of lung biology, immunology, and mechanisms of injury and repair.84,91 Beyond the day-today diagnostic and therapeutic applications, FB has been used as an investigative tool for specimen retrieval, most widely in normal volunteers but also in a variety of chronic interstitial lung diseases including sarcoidosis, idiopathic pulmonary fibrosis, hypersensitivity pneumonitis, pneumoconiosis, histiocytosis X, and drug-induced lung disease.40,42, Historically, airway diseases such as asthma and emphysema were considered relative contraindications for FB. In the past 10 to 15 years, investigative bronchoscopy has been extended to asthma and other airway disorders.63,96, lI7, 196 The result of the study of asthma with FB and lavage has been a dramatic shift in our understanding of asthma, the impact of which must be considered at least as significant as the impact of FB in the research of interstitial lung disease.”, 43, A number of reasons exist for the increasing use of investigative FB for asthma, the most important of which is a gradual and cumulative record of safety.25, 94, lo7 In addition, a coalescence of technologi-

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cal advances in a number of fields have contributed including: ability to perform multiple mediator assays,166advances in molecular and cytokine biology (e.g., use of techniques such as polymerase chain reaction to assay mRNA transcripts for particular mediators from lavage cells), and advances in challenge procedures such as whole lung aerosolized allergen challenge or segmental allergen challenge (SAC) followed by serial bronchoscopies to investigate the kinetics of inflammation at several time points.27 The major advance of investigative FB with lavage has been the ability to obtain information that is not readily available by other monitoring methods in asthmatics. In addition, recovery of viable and functioning inflammatory cells and the ability to quantify this inflammation (cellular and noncellular) offers new avenues to study. Information from the investigative FB in asthmatics has largely contributed to the evolving paradigm 53 Beof asthma as an inflammatory di~ease.~, fore the use of FB, autopsy studies of patients who have died with acute fulminant asthma did suggest widespread mucosal inflammation. Investigative FB, however, has provided a tool to study patients with milder disease, patients in remission, and a variety of models of experimentally induced asthma in vivo. This information has strongly supported the

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From the Pulmonary Function Laboratory (MSK), Department of Pulmonary and Critical Care Medicine (RAD, MT), The Cleveland Clinic Foundation, Cleveland, Ohio

CLINICS IN CHEST MEDICINE

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VOLUME 20 NUMBER 1 MARCH 1999

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inflammatory hypothesis, that is, airway epithelial and mucosal inflammation is associated with airway hyper-reactivity and is present even in the mildest asthmatics and in asthmatics in remission.131In addition, sophisticated multiple mediator studies have given some insight into the role of a variety of cells and soluble mediators, including eosinophils and TH,-lymphocytes and a variety of proand anti-inflammatory cytokines. This article reviews the technique of FB in asthma research. The article is divided into the following sections: general guidelines from several workshops and the relative safety of FB; technical issues related to specimen retrieval and processing; and data obtained from studies that have used bronchoscopy for investigation of stable asthma, asthma with allergen provocation, or asthma treated with anti-inflammatory therapy. This article is primarily confined to asthma research applications rather than therapeutic applications (e.g., BAL as therapy in status asthmaticus). Only adult human studies are considered. The discussion is limited to the technique for the lower airways and issues related to nasal lavage and nasal allergen challenges are not covered. Finally, the current state of knowledge, limitations, and future directions of FB in asthma research is summarized.

TECHNICAL ISSUES Over the past 25 years, BAL by way of FB has been used as a research tool. The goal of much of the research has been to define cellular and noncellular components of normal lung and disease states. Despite several workshops, BAL has been performed by a variety of technique^.'^, l4 The following lists summarize indications and some guidelines for investigative FB.

Indications for Investigative Bronchoscopy in Asthma: 1. Obtain fluids, cells, and tissue samples to study morphology histology molecular biology immunology biochemistry pharmacology 2. Study local airway physiology 3. Evaluate bronchial blood flow

Evaluate the effects of therapeutic intervention Study airway epithelium in situ and in vitro Study pathogenesis of airway infection Measure regional airway gases (e.g., nitric oxide)

Summary of Recommendations and Guidelines for Investigative Bronchoscopy in Subjects with Asthma: Contraindications Absolute sensitivity to local anesthetic uncorrected bleeding diathesis (especially if biopsy is planned) history of status asthmaticus Relative FEV, less than 60% predicted unstable cardiovascular disease upper respiratory tract infection less than 4 weeks age greater than 60 years Preprocedure evaluation medical history, examination current medications, allergies blood pressure, pulse, intravenous access spirogram, pulse oximetry premeds: atropine, sedation, 4 p-aerosols resuscitation facilities During procedure evaluation continuous oximetry, blood pressure, EKG monitoring topical lidocaine total dose less than 400 mg supplemental oxygen terminate procedure if bronchospasm occurs Postprocedure evaluation observe until: gag reflex returns no significant airflow obstruction discharge instructions call if fever, chest pain, drop in peak flow phone follow-up Specific Procedures bronchoalveolar lavage total lidocaine less than 400 mg use normal saline at 37°C endobronchial brush limit to 2 to 4 areas endobronchial biopsy less than six 2 mm biopsies allergen provocation use lowest dose necessary to study research issue (by prior skin, aerosol testing) use endotoxin-free extracts

ROLE OF BRONCHOSCOPY IN ASTHMA RESEARCH

A number of issues have been actively studied by investigators primarily in the normal lung: (1) What is the ideal location for sampling, and what is the optimal volume of the instillate? (2) What are the differences between "bronchial" versus "alveolar" samples and what are ways to enrich the bronchial sample? (3) What can be learned from sequential lavage at multiple time points? (4) What is the relationship between BAL and other compartments such as airway brushings and biopsy? (5) What is the impact of research bronchoscopy on lung function, airway hyper-responsiveness, and lung inflammation? and (6) What is the overall safety of this technique? These issues are reviewed in this section. Advancing an 8-mm bronchoscope in the airway usually results in wedging to occur between the 4th and 6th order bronchus.91 Figure 1 shows a schematic of investigative bronchoscopy methods to obtain airway specimens. Wedge position typically localizes a lung zone with lo6 alveoli, or a volume of 165 mL at total lung capacity and 45 mL at residual volume. The procedure is generally performed with the patient in a supine position in an outpatient bronchoscopy suite with light sedation. The yield is generally lower

155

when this technique is performed under general anesthesia. de Blasio et a1 studied the impact of local anesthesia, general anesthesia, and mechanical ventilation without general anesthesia on BAL findings in a large number of patients.45 They found a significantly higher fluid recovery with local anesthesia. The yield was similar for the other two groups, suggesting that the reduced fluid return is related to mechanical ventilation rather than anesthesia. Pingleton studied the effect of location on BAL in normal Percent fluid recovery was greater in the right middle lobe than in the lingula and greater in the middle lobe and the lingula than in the lower lobes. Total cell count was significantly higher in the right middle lobe than in the left lower lobe. Cell count per mL and protein recovered, however, were not different between any lobe lavaged. The weakness with this study is that the lavage was performed with the patient in a seated position, which is not the usual practice. Although it is generally stated that upper lobes have a lower yield, the anterior subsegments of the upper lobes may be acceptable.70,147,195 Sterile normal saline (0.9% NS, with or without buffering) is infused through the suction port. Saline

Figure 1. Technique for obtaining cellular and noncellular components during investigative flexible bronchoscopy (FB). Specimens obtained from FB may include bronchoalveolar lavage (BAL), mucosal brushings, endobronchial biopsy, or airway gases (e.g., nitric oxide). By centrifugation, BAL is separated into fluid supernatant or cells. After total and differential cell count, cells may be placed in culture or extracts prepared for polymerase chain reaction (PCR). Fluid may be concentrated for additional assays.

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warmed to body temperature of 37°C seems to have increased return over room temperature, especially in patients with asthma.138In various studies, fluid instillation has been anywhere from 10 mL to 100 mL per aliquot repeated from 3 to 6 times for a maximum volume of 300 to 600 mL. Most studies have placed the scope in a wedged position, although some studies have looked at an unwedged collection of bronchial washings compared with a wedged collection. The fluid is typically recovered by gentle hand aspiration with a syringe by way of the suction port; sometimes aspiration by way of wall suction at 50 to 100 mm Hg is used, but this approach is more traumatic to the airway and is not preferred., Investigators have withdrawn the fluid either immediately after instillation or after some ”dwell time.” Longer dwell time seems, within limits, to increase the overall cell yield. A multicenter study evaluated BAL fluid constituents in normal volunteer^.^ This study evaluated 191 normal volunteers by a BAL technique involving 60 mL aliquots X 4 at room temperature. An analysis was done on the pooled recovered volume. The mean recovery of BAL fluid decreased with increasing age. Also, the recovery of BAL fluid was significantly less in current smokers compared with never smokers. Fluid recovery in African Americans on average was 10 mL (4.3%) less than white individuals. After adjusting for volume recovered and smoking history, there was no effect of age, gender, or race on BAL fluid cell populations. Total cells and cells/mL in BAL fluid were three times as high in current smokers as in nonsmokers. The number of macrophages and neutrophils was 4 to 6 times higher in smokers than in nonsmokers. A variety of studies from normal volunteers indicate that the volume of recovery varies between 40% and 60% of the instilled volume (Table 1).In normal nonsmokers, the mean values for total cells range between 5.9 and 22.2 million (from 58,000 to 269,000 cells/ mL), the mean values for macrophages are 86% (range 70% to 95%), lymphocytes 10% (range 4 to 18), neutrophils 1.7% (range 0% to 12%),and no eosinophils. In current smokers, the mean number of cells ranged from 14.4to 81.7 million (335,000 to 1.15 million cells/ mL), whereas the mean percentage of neutrophils varied from 0% to 8% and that of lymphocytes from 3% to 8%. Cell viability, as assessed by trypan blue dye, is typically 92%

in current smokers and 86% in nonsmokers. Lymphocyte surface antigen phenotyping in BAL in normal nonsmoking subjects consists of 9.5% lymphocytes; of these 71% are T cells (41%were T helper cells, 23% were T suppressor cells with a TH/Ts ratio of 2.3) and 4% were B-lymphocytes. The normal blood TH/ Ts ratio is 2.2. In smokers, the peripheral blood T-helper cells increase and the TJT, ratio increases from 2.1 to 2.3. In BAL of current smokers, however, there is a decrease in T-helper cells and the TH/Ts ratio is reduced. In normal subjects the total protein is between 1 and 10 mg, or 89 mcg/mL, being mostly albumin (40.2 mcg/mL), IgG (8.2 mcg/mL), and IgA (6.5 mcg/mL).21 Ettensohn et a1 performed repeated BAL in normal subjects to assess variabilityF A total of 59 BALs were performed in 16 volunteers (3 aliquots of 40 mL at room temperature in a lingular subsegment). BALs were performed on different days with a minimal interval of 6 weeks. Subjects underwent between 3 and 5 repeated BALs. Data indicated significant variability in percent fluid return, number of total cells, and cells per mL BAL fluid. The cell differential was the most consistent parameter on repeated BAL. The authors concluded that some of the variability may have been caused by different subsegments within the lingula being sampled on different occasions. Performance of prior BAL did not consistently either increase or decrease the numbers. Finally, this study demonstrated that lung function is not significantly affected by repeat BALs and the overall safety of FB is acceptable. Some investigators have suggested that the variability in lavage return could be caused by residual pockets of air in a lavaged segment that would lessen return. Carre, in an animal model, illustrated that atelectasis or “degassing” a segment increases BAL return and cell~larity.~~ Degassing, however, has no role in human studies. A number of studies looked at the impact of lavage volume.44,55, 83, lo9 Davis et a1 performed BAL with 60 mL aliquots four times in the right middle lobe.@Each aspirated aliquot was separately analyzed as well as a combined ”pooled” sample. They noted that between the first syringe and the fourth syringe the recovered fluid increased from 28 mL to 47, 51, and 60 mL. The total cell yield in absolute numbers increased between syringe 1 and syringe 2 and then stayed fairly constant, whereas cells/mL were actually the highest in the first syringe and subsequently

100 Unknown 150 100 to 300 150 Unknown 240 120

7 26 8 42 16 10 6 78

1975 1976 1977 1977 1978 1978 1979 1981 1983 1983

1983 1984 1984 1985 1986 1986 1987 1988

74 78 79 78 70 93 95 84 90 89 93 93 86 88 89 92 86 89 95

NR 12.7 13.9 13.1 17.6 10.2 6.4 15.3 15.7 11.0 26.9 13.0 22.5 16.3 5.8 NR 13.0 NR 9.4

9.5 22.2 6.3 18.9 5.9 11.2 NR 16.0 14.6 14.0 9.4 14.9 15.1 NR NR 15.7 16 7.3

NR NR 0 NR 0 0 NR NR NR 0 1 1 0 0 NR NR 0 0

NR NR 0 12 1 2 NR 3 1 0 0 1 1 2 1 NR 1 1

17 17 18 8 7 6 11 7 8 7 6 12 11 10 7 8 10 4

0

("a

Eosinophils

0

Neutrophils (%)

15

("/I

(%I

12.8

Lymphocytes

Macrophages

Cells/ mL ( x 104)

Total Cells ( x 108)

NR = not reported. From BAL Cooperative Group Steering Committee. Bronchoalveolar lavage constituents in healthy individuals, idiopathic pulmonary fibrosis, and selected comparison groups. Am Rev Resp Dis 141:169-202, 1990; with permission.

63 63

63 69 NR

69

50 NR 48 43 45 73 51 68 NR 52

100 to 200 250 100 250 100 240 <300 150 NR 100

12 24 11 10 10 14 16 12 7 8

1974

Reynolds and Ne~bali'~~ Daniele4I Warrlw ReynoldsIM YeagerZ1O WeinbergerZw

DaubeP3 Belllo DeShazo50 Hunninghake and Crystalgo Pinglet~n'~~ YasuokaZW Vell~ti'*~ LaViolette1I2 Baughman7 WeissmanZw Morgan'29 Ettensohn6I

66

150

5

Year

Percent Recovery

Author

Volume Instilled

Number of Patients

Table 1. CELLS IN BRONCHOALVEOLAR LAVAGE FLUID IN NORMAL NONSMOKING SUBJECTS

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KAVURU et a1

declined in nonsmokers. Noncellular constituents generally appeared in declining concentrations in sequential syringes. Overall, it seems that a simple mixing model does not fully explain the findings. Merrill et a1 did a similar study of a BAL in the right middle lobe of 14 normal volunteer^.'^^ Sequential 50 mL aliquots were administered for a total volume of 300 mL. They noted a progressive decline in the concentration of all proteins from the first aliquot to the sixth aliquot. There, however, was no change in proteins adjusted to a reference value such as albumin or total protein. Investigators have advanced two possible theoretical models to help understand the recovery of proteins from lung during lavage 196 One model suggests with serial aliquot~.'~~, that lung distal to the wedged bronchoscope is comprised of conducting airways terminating in alveolar spaces in series. The lavage method preferentially samples airway protein in early aliquots and alveolar proteins in later aliquots. This hypothesis would imply greater concentration of airway specific proteins (relative to albumin or total protein) in early versus late lavages. The second model suggests that lung distal to the bronchoscope tip represents a homogenous pool of soluble protein that is progressively diluted as protein containing fluid is aspirated and further aliquots of normal saline are instilled. This model would suggest a progressive decrease in protein concentration from aliquot to aliquot but no alteration in calculated protein ratios. A study by Merrill et a1 seems to favor the second hyp~thesis.'~~ Ward et a1 examined intrasubject variability of cellular and solute parameters in repeated 180 mL BALs in 20 clinically stable but symptomatic asthmatic^.'^^ The authors noted variability and expressed it in terms of a standard deviation (SD) of the differences of repeated measurements: total cell count SD is 78 X lo3 cells/mL, macrophages 16%,lymphocytes 13%, eosinophils 1%,and albumin 40 mcg/ mL. The authors formulated a sample size estimate of 15 patients in each group for studies of mild-to-moderate symptomatic asthmatics using bronchoscopic endpoints of BAL and endobronchial biopsy. Many attempts have been made to determine what proportion of the recovered fluid volume from a BAL represents the fluid that lines the epithelial surfaces of the distal airways and alveoli, often referred to as epithelial lining fluid (ELF). Several methods have

been suggested to control this problem of variable dilution: (1) Present data as a ratio of the concentration of different proteins of interest to the concentration of total protein or albumin; (2) Ratio of protein concentrations to concentration of potassium; (3) Add a low concentration of methylene blue to the instilled saline so a dilution factor can be derived from the difference in the concentration of methylene blue in the instilled and aspirated lavage fluid and from this the ELF can be calculated; and (4) Determine the volume of ELF by a ratio of serum urea to BAL urea, because low molecular weight urea is believed to be highly permeable, will cross membranes, and the concentration of urea in ELF should be equal to that in plasma. Volume of ELF recovered from BAL can be calculated as follows"?

Concentration of albumin in ELF:

Kelly et a1 studied fluid dynamics during BAL by the use of methylene blue, technet i ~ m colloid ~ ~ tracer, and tritiated water.lo0,lol These experiments suggested that a bidirectional flux of water occurs across the alveolar membrane with a significant net influx of fluid from the circulation into the lung and that less than 2% of the total aspirated volume comes from fluid present in the lung before BAL (Fig. 2). The same authors used radio-opaque dye and digital subtraction radiography to visualize the anatomical distribution of 60 mL aliquots X 3 introduced sequentially into the right middle lobe.'O1 They noted that the first 60 mL aliquot stayed close to the bronchoscope and probably sampled only the proximal airways. With the subsequent introduction of 120 mL the fluid filled the segment more evenly and aspiration then moved fluid back from the periphery implying that the second aspirate of the lavage samples the distal airways and alveoli. Several studies have found limitations with attempts to quantify ELF. Marcy et a1 calculated the ELF in six healthy nonsmoking subjects using two different BAL protocols, 5 X 20 mL and 6 x 50 rnL."* The urea concentration increased progressively from the first to the sixth aliquot, suggesting that a significant amount of urea diffuses into the instilled BAL fluid as it dwells in the alveolar space. This

ROLE OF BRONCHOSCOPY IN ASTHMA RESEARCH

-1

1-

Bronchopulmonary segment

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0 CCF 1998

Figure 2. A bronchopulmonary segment depicting complex fluid movement during a typical research bronchoalveolar lavage (60 mL x 3). From a total injectate of 180 mL, approximately 60 mL effluxes into the interstitium or vasculature and 110 mL influxes from the vasculature secondary to aspiration pressure. The total dilution volume = 180 mL + 110 mL - 60 mL = 240 mL. If 100 mL is aspirated back, it is comprised of 60 mL from injectate, 40 mL from influx from blood, and less than or equal to 2 mL from epithelial lining fluid (ELF). (From Walters EH, Ward C, Li X: Bronchoalveolar lavage in asthma research. Respirology 1:235, 1996; with permission.)

diffusion of urea causes errors in the calculated ELF recovery. Von Wichert et a1 performed a comparative study with five markers to attempt to quantify ELF.188They noted that in humans, calculation of ELF with the urea method and with technetium showed no correlation. Helmers et a1 studied the effective volume of fluid infused in interstitial lung disease.83The study was designed to perform three BALs in one procedure; a 100 mL BAL and two 50 mL BALs were performed in two adjacent segments of the lingula and a 100 mL BAL from the right middle lobe. The percent of fluid return and the total number of cells was greater in the two 50 mL versus the 100 mL lavage. There, however, were no significant differences in cell differentials or numbers of cells per mL between the volumes in these patients with interstitial lung disease. There have been a number of attempts to modify the BAL technique to selectively enrich the bronchial sample to study airway disease such as asthma, in contrast to the alveolar sample that is more suitable for interstitial lung disease. These techniques have included: (1)Small volume unwedged lavage

of large ainvays.lo5This typically has a return of 1 to 10 mL and typically more than one lavage per side cannot be performed. (2) A small volume lavage through a wedged bronchoscope. (3) Multiple sequential lavages. (4) Lavage of segments isolated by a balloon catheter.212Balloon lavage yields a specimen rich in ciliated epithelial cells of approximately 50%.212This technique, however, is cumbersome, could only sample the large proximal airways rather than the distal airways, and the return is usually quite variable. (5) Finally, fractional processing of BAL was proposed by Rennard et al.'" This technique involves separate processing of fluid return from the first aliquot of 5 to 20 mL ("bronchial specimen") infused into a single segment through the wedged scope, whereas additional aliquots are po01ed.l~~ The volume recovered and the total number of cells is smaller in the bronchial specimen. Bronchial specimen apparently has increased numbers of columnar epithelial cells, polymorphonuclear cells, and IgA. The contribution of cells and differential to the pooled BAL specimen is small by comparison with the bronchial

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KAVURU et a1

specimen. Fractional processing in normals This includes the syringe for suctioning, tubshows a significant difference between the ing as well as suction trap (if this were to bronchial specimen and the pooled specimen. be used), and the container for transport. In This, however, may be much less important general, total cell counts performed on lavage in patients with airway disease. fluids are most accurate when determined Several investigators have attempted to asfrom the original, uncentrifuged, pooled specsess airway inflammation by a visual endoimen. Each step in processing leads to cell scopic scoring system in patients with asthma To remove loss and decrease in ~iabi1ity.l~~ and chronic bronchitis.8,178, The score demucus debris samples are filtered through scribed by Thompson et al, the bronchitis inloose gauze (loose mesh nylon is preferred dex, is a scale that is graded from 0 to 3 for over cotton gauze). Cell counts may be carerythema, edema, secretions, friability, with ried out with a coulter counter or a hemocyo0 = normal, and 3 = severely abn0rrna1.l~~ meter. Cytologic examination requires conThe index is determined by adding all the centration of cell contents. Several techniques scores from six sites and thus the grading have been used: whole specimen cytocentrifurange is from 0 to 72. A second scoring sysgation, membrane filtration, cover glass seditem was proposed by Vyve et al, "endoscopic mentation, or pooled glass cover preparation.lll, 179, 193 score": hypersecretion, hyperemia, edema, and friability are graded from 0 (absence) to The cytocentrifugation is a technique that 1 (presence) and this refers to a score aris used most often as it is quick and reproducranged from 0 to 4.ls9These two scoring sysible and requires a small specimen (< 0.5 mL tems were compared in 60 asthmatics of varior 50 to 100,000 cells per smear). Centrifugaable severity and 30 healthy controls who tion is typically performed at 400 g for 6 to subsequently underwent b r o n c h o s c ~ p y . ' ~ ~10 minutes. For a bloody specimen the red The macroscopic examination of the airways cells are lysed with a hypotonic solution. With 'was performed by two independent observthis technique, percentage lymphocyte^'^^ are ers and there was a significant correlation consistently underestimated, representing a between the two endoscopic scores in asthmaloss between 25% and 50% of lymphocytes. tics and in controls. Also, there was a correlaMembrane filtration or millipore filter prepation between the endoscopic score and the ration is most time consuming, typically reclinical score, which was generated based on quires 100 to 200,000 cells per smear, and symptoms, medication use, and peak flow preserves nuclear detail well but underestimates neutrophils. Specimens are stained by a measurements. There, however, was no correvariety of techniques for differential counting lation between the endoscopic scores and the BAL cell differentials. Currently, there are inincluding Wright-Giemsa or modified Wrightsufficient data to determine whether a visual Giemsa (Diff Quik), or Papanicolaou method. endoscopic score provides any unique inforThe BAL supernatants can be stored at 4°C mation beyond either clinical or physiologic and assayed for various proteins within a few parameters or quantitative parameters from days or more likely be frozen at -70°C for BAL or biopsy. future batch processing. The fluid can be concentrated by negative or positive pressure ultrafiltration. The normal human BAL typiSPECIMEN PROCESSING cally does not contain proteins with molecular weight greater than 300 kilodaltons. MemBronchoalveolar Lavage brane filters may be used to obtain low molecular weight proteins. Concentration After recovery of the instillate, a variety of methods typically lead to loss of protein, technical factors may influence the total cell which adheres to the filter. Proteins can be count and differential.s5The fluid should be assayed by radioimmunodiffusion, radioimcollected and transported to the laboratory in munoassay (RIA), or enzyme linked immunocontainers made of materials to which the sorbent assay (ELISA). cells are poorly adherent.84 Unsiliconized glassware has been used in the past and this Bronchial Brushings and Biopsy significantly lowers cell recovery. Disposable Since the early studies evaluating tracheoplasticware made out of material such as polyethylene or polycarbonate is preferred. bronchial biopsies obtained through the rigid Siliconized glass materials are also acceptable. bronchoscope, there have been increasing

ROLE OF BRONCHOSCOPY IN ASTHMA RESEARCH

161

Table 2. ADVANTAGES AND DISADVANTAGES OF THE DIFFERENT TECHNIQUES FOR PROCESSING OF BRONCHIAL BIOPSIES

Advantages

Histochemistry Immunochemistry Paraffin Araldite Glycol methacrylate

Disadvantages

Good morphology Rapid staining suitable for sample orientation and preliminary overview

Bad molecular characterization of different cell types

Good morphology Immunoreactivity for increasing number of antibodies Good morphology Thin sections

Not all antibodies react

Frozen

Good morphology Thin sections Immunoreactivity for increasing number of antibodies Immunoreactivity for all antibodies

Electron microscopy

Good morphology Identification of ultrastructural changes

Few reacting antibodies Expensive instruments for cutting ultrathin sections Expensive instruments for cutting ultrathin sections Bad morphology Limited duration of immunoreactivity Expensive instruments Long procedures

From Saetta M, Jeffery PK, Maestrielli P, et al: Biopsies: Processing and assessment. Eur Respir J Suppl 2620%25S, 1998; with permission.

numbers of studies using FB to obtain endobronchial biopsies for investigational purposes in asthmatic volunteers. Advances in histochemistry and immunohistochemistry techniques have enabled investigators to study the small specimens obtained by endobronchial and transbronchial biopsies. Table 2 summarizes the advantages and disadvantages of the different techniques for processing bronchial biopsies. More recently, techniques have been developed to harvest viable tracheobronchial epithelial cells from normal and asthmatic volunteers.60Studies suggest that airway epithelial cells may play a significant role in asthmatic airway inflammat i ~ n .49,~59,~lS5 , Brushing the mucosal surface of the trachea and the mainstem bronchi through FB yields between 10 and 20 million cells. The three major cell types present in the mucosa include ciliated, secretory, and basal cells, and all are usually obtained.'02 The major advantage of mucosal brushings is that harvested cells may be placed in primary cell culture for in vitro studies with a high plating efficiency. USEFULNESS OF INDUCED SPUTUM AND BRONCHOALVEOLAR LAVAGE IN ASTHMA Induced Sputum A number of investigators have recently used a noninvasive technique to assess air-

way inflammation, mostly in patients with mild asthma compared with healthy controls. It is generally believed that spontaneously produced sputum is inadequate and that induced sputum is required to obtain an adelz2 A sumquate specimen for processing.104, mary of the published techniques for sputum induction and processing follows: Pretreat with inhaled p,-agonist Select subject with mild-moderate stable asthma (FEV, > 60% pred.) Inhale aerosolized hypertonic saline Aerosol generated by ultrasonic nebulizer over a fixed time (e.g., 20 to 30 minutes) with saline concentration of 3% or 4% or 5% OR Inhale aerosol of same concentration (4.5%) over increasing time periods High frequency oscillator does not increase sputum volume or cell yield After every 3 to 5 minutes (or a change in aerosol concentration), cough and expectorate Rinse mouth and blow nose before cough Monitor serial FEV, Sample processing Select mucus plugs from sample OR Process whole expectorate (sputum plus saliva) Liquefy sample by adding dithiothreitol (DTT) 0.1% to disperse cells, stain cytospins

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Report cell viability, YO squamous cell contamination, total cell count and differential Sputum supernatant for measurement of soluble mediators

fluid-phase levels of eosinophil chemotactic protein (ECP), major basic protein (MBP), eosinophil derived neurotoxin (EDN), and interleukin-5 (IL-5) than the other two groups. Veen et a1 studied the reproducibility of cellular and soluble markers of inflammation in Induced sputum has been compared with sputum induced by hypertonic saline in 12 patients with mild, atopic asthma without inbronchial washings and BAL.&,78, 90, 116 A1so, studies have used induced sputum to demonhaled steroid treatment and 9 patients with strate changes in the cellular constituents and moderate to severe atopic asthma treated soluble mediators with aerosolized allergen with inhaled steroids on two separate days at 137 In addition, the effects of prov~cation.~~, least 2 days aparf.ls4Whole sputum samples steroid therapy on induced sputum have been were induced by inhalation of 4.5% saline; evaluated in asthmatics. Although it is known they were homogenized and analyzed. The that hypertonic saline can have a bronchoconreproducibility expressed as intraclass correstricting effect, most studies have shown lation coefficient was satisfactory and ranged overall safety for this technique in patients between 0.60 and 0.85 in the two groups comwith well controlled mild asthma. In fact, the bined. For eosinophils, the coefficient was mean fall in FEV, is 5% to 7% from baseline high at 0.85. This study supports processing (range of 0% to 47%) with sputum induced of induced sputum by using the whole expecby hypertonic saline. One recent study even torated sample without further selection or described this technique in patients with seseparation. vere unstable asthma as ~ e l l . 4de~ la Fuente Four studies have critically compared instudied the safety of hypertonic sputum induced sputum with bronchial washings and duction in 64 asthmatic patients with varying BAL in asthmatic and healthy subjects (Table severity of disease including 21 patients with 3). Fahy et a1 found that sputum, compared uncontrolled asthma.47The procedure was with either bronchial washings or BAL had well tolerated in most patients but had to higher numbers of non-squamous cells, be stopped because of side effects in 11.6% higher levels of eosinophil cationic protein, patients with severe asthma.47A drop in FEV, and albumin.65Also, there is a much higher of 10% to 20% occurred in 18% patients with percentage of neutrophils in the induced spusevere asthma. Reports suggest that adequate tum specimen. The eosinophil percentage and specimens can be obtained in more than 75% the ECP level in sputum correlated more of all attempts. It seems that the hypertonic closely with those in bronchial washings than saline itself does not induce airway inflamin BAL. These authors concluded that the mation or affect the profile of cells compared analysis of induced sputum reveals informawith normal sa1ine.l” tion qualitatively similar to that obtained by Several studies have noted that the methanalysis of washings and BAL. Maestrelli et ods for measuring cell and fluid phase marka1 also found that the percentage of neutroers in induced sputum are reproducible and phils was significantly higher in sputum than valid.140They may be used reliably to meain BAL in stable asthmatics and that macrosure indices of airway inflammation. Pizziphages and lymphocytes were higher in the chini et a1 examined the reproducibility and BAL and bronchial submucosa.116Also, BAL validity of sputum induced by hypertonic saeosinophils correlated with sputum eosinoline on two separate occasions within 6 days phils and submucosal eosinophils in patients in 10 healthy subjects, 19 stable asthmatics, with asthma. Keatings et a1 also studied inand 10 smokers with chronic b r 0 n ~ h i t i s . l ~ ~duced sputum and bronchoscopy with bronFreshly expectorated sputum was separated chial washings and BAL in random order, from saliva, was treated with a proportion with each procedure being separated by an of dithiothreitol 0.1% followed by Dulbecco’s interval of 12 days in 16 patients with mild, phosphate buffered saline, making cytospins stable asthma.99Again, induced sputum was and collecting the supernatant. Overall, the relatively rich in neutrophils and eosinophils intraclass correlation coefficient was high for compared with washings or BAL. Finally, all indices measured with the exception of Grootendorst et a1 studied 18 clinically stable total cell counts and proportion of lymphoatopic asthmatics by sputum induction and c y t e ~ .Asthmatics ’~~ had higher proportions of bronchoscopy on separate days in random order.78The percentage eosinophils in sputum eosinophils and metachromatic cells and Texf continued on page 168

TCC = total cell count; IS

Number of Asthmatics Specimen Source TCC, 1 0 5 / m ~ Epi Cell % Macro % PMN Yo EOSYo Lymph % ECC ng/mL Albumin, mcg/mL

=

BW 1.6 4.2 90 1.9 0.9 1.8 0.4 43

10

Fahy (1995)65

10 BAL 2.8 0.3 93 0.8 0.2 2.9 0.1 53

13 BAL 75 6 8 8

44 40 14 0.5

=

85 5 1.6 0.2

88 1.3 0.6 11.8

14 BAL

eosinophilic cationic protein

60 36 3.3 0.3

150/ mmz 48/mm2 133/mm2 448/mm2

14 BW

Keatings (1997)99

14 IS

21 Biopsy

Maestrelli (1995)116

13 IS

induced sputum; BW = bronchial washing; BAL = bronchoalveolar lavage; ECP

10 IS 4.6 1.1 30 36 1.9 0.4 77 205

X

17 BW 1.2 x 106 5.5 7.5 2.3 1.8 10.6

18 BAL 8.8 X lo6 0.2 87 1.4 1.2 9.7

Grootendorst (1997)78

lo6 6.9 49 29 1.0 3.3

4.8

IS

18

Table 3. COMPARISON OF INFLAMMATORY CELL COUNTS IN ASTHMA BY SPECIMEN SOURCE: INDUCED SPUTUM, BRONCHIAL WASH, OR LAVAGE

RML 6 0 x 3 (warm)

RLL5OX2

Flint6' (1985)

Lamlw (1985)

K i r b ~ ' ~ ~ RML/ Liigula (1687) 20x5

RML

Location of BAL

Godard" (1982)

Author (year)

10

14 17

NV 7

100

10

63

45 69

100 100

68

63

45 69

40 50

38

3.6 (5.7)

8.9 (19.9) 4.4 (6.35)

11.7 (16.3) 7.2 (14.4)

12.2 (17.9)

19)

(15.5

N/S 2

(2.9 f 20)

%

Total Number of Cells (lo6)or (10' celldmL)

Out N/S

13

100

300-400

In 300-400

72 50

NA-A

Volume (mL)

180 100

10

15

ASA-ASX

Patient Subgroup

16 5

6

82

8 12

9

14

60 90

86 78

80

80

3

3 2

4 2

3

1.4

P 0

Differential

Table 4. BRONCHOALVEOLAR LAVAGE FOR CHRONIC STABLE ASTHMA: SUMMARY OF PUBLISHED STUDIES

1

4 0

2 0

8

4

Eos 0.4

2

6 3

5

Epi

Mild (1.34)

Western red cedar asthma

MildModerate Mild

Small volume lavage recovered more epis, PMNs, while volume 25 to 100 mL recovered more lymphs, AMs, albumin.

Comments

P-MDI MBP in BAL & <8 h. blood did not No other differ for the 2 groups. meds. Eos and metachromatic cells were in BAL and bronchial wash (first 25 mL) of asthma. Bronchial washing did not distinguish asthma and normal any more than BAL.

N/A

No P-MDI 2 weeks

8 days off therapy

Asthma Severity Rx (PC, mg/mL) Withhold

9

2 M U ?

d

\

M

-?

2

0 3 M

m

m

N

Lo

D

0 3 3

N

m m a

b

9

m h m

m

mu,

9

w

c

L

m

0

z :

3b

r!

2

o

o

r

.

Lo

c:

o

o\ Lo

0

0

2

2

s

2 m

3

00

13 3

m

o

3 N

N

3

\

D

4

&

165

M

RML 50 x 3

Walker19' (1991) Walker192 (1992)

RUL 5 0 x 5 (room temp)

RML50X5 (room temp)

Wilson*" (1992)

VyveIqo (1992)

RML 5 0 x 3

Location of BAL

Author (Year)

31

10

10 10

NV

AS

12

44

120

96

200

36.6 59.9

out

Volume (mL)

200

200

150 150 200

In

10

10

NA-A 150 150 150

56

SX

17

A-A

Patient Subgroup

48

14.7 (15.3)

17.5 (14.6)

82.0

86.1

59 60

92

N/A

70

85.5 88.3 88

83.6 83.7 94

9.7 (26) 8.2 (13:7) 8.23

MP

9.0 6.4 N/A

25 40

Yo

Total Number of Cells (lo6)or (lo4 celldrnL)

10.1

10.5

4

13.3 11.1 5

11.7 9.4 10.1

L

2.9 3.7 0.4 2.6 2.7 1

2.9

0.3 1.7

2.7

4.3

1

1.3

2.0 0.2 0.2

Eos

1.3

1.8

P

Differential

Table 4. BRONCHOALVEOLAR LAVAGE FOR CHRONIC STABLE ASTHMA: SUMMARY OF PUBLISHED STUDIES (Continued)

2.6

2.5

Epi

Comments

MildModerate (0.61) MildModerate

Pred >8 weeks, P-MDI >8 h

Pred >8 weeks

bronchial, alveolar sample.

tin

Bronchial sample (first 50 mL) contained more PMNs, eos, fewer macros, lymphs, than pooled alveolar sample (in NV, asthma), Alveolar sample contained more cells/mL in NV, asthma, In asthma, epis

No difference in ratio CD,, CD,, (-2.5)or

AA only, 1.92/ 1.6, AA + t IL-4, IL-5, NAA + t IL-2, IL-5, 2.79/1.1

Moderate 1.2 P-MDI, IC t IL-4 in AA in blood BAL >12 h, (AA), 0.9 (NA-A) Pred T-cells, >3 Blood/BAL CD4/CD8 = months 1.8/ 1.7, t IL-4/IFN-y in

Asthma Rx Severity (PC,, mg/mL) Withhold

60x4

RML/ Lingular

(warm)

60x3

RML/ Lingular

10

13

10

10

19

240

240

180 180

180

130

115

70

63

(28)

11.7 (9)

11.5 (10)

93

85.3

84.5

2.9

14.3

11.3

0.8

0.4

0.5

0.2

0.4

1.8

0.3

0.2

0.1

(0.6) ('32)

Moderate

Mild (10.4)

Pred, IC >6 weeks

N/A

Induced sputum is more concentrated in cells, ECP, alb, higher YO PMNs, more closely resembles bronchial sample than BAL.

I

CD,, expression by CD, T-cells was greater for asthma in blood, BAL; BAL CD, T-cells from asthma were of memory phenotype (CDG RO + R.-).

NV = normal volunteer; A-A = allergic asthmatic; NA-A = nonallergic asthmatic; AS = asymptomatic; Sx = symptomatic; RLL = right lower lobe; RML = right middle lobe; RUL = right upper lobe; Mp = macrophage; L = lymphocyte; P or PMN = polymorphonuclear leukocyte; eos = eosinophil; epi = epithelial cell; P-MDI = Beta-agonist metered dose inhaler; IC = inhaled corticosteroid; t = increased; BAL = bronchoalveolar lavage; MBP = major basic protein; ECP = eosinophil cationic protein; GM-CSF = granulocyte macrophage colony stimulating factor; IL = interleukin; N/S = not specified.

(1995)

Fahp5

(1993)

RobinsodW

168

KAVURU et a1

was significantly correlated with their percentage in bronchial washings and in BAL. Also, there was no significant difference in sputum cell differentials in patients on or off inhaled corticosteroids. Additional studies assessed a variety of mediators and found that induced sputum combined with polymerase chain reaction to detect messenger RNA for a range of cytokines on a qualitative basis is effective. In summary, the emerging data on the use of sputum induction to study patients with asthma seems prornising.‘j8The main advantage is that this technique is not invasive, is technically much easier and requires less expertise and may be sequentially repeated multiple times. Another advantage is that the induced sputum specimens seem to be fairly concentrated and they do not have the problem of variable dilution from the instillate as does BAL. It seems that the induced sputum specimen mostly represents specimen from large airways and that the technique itself does not seem to produce inflammation. It has been consistently shown that the induced sputum specimen is rich in neutrophils, eosinophils, and eosinophil products in much higher concentrations than in bronchial washings or BAL. Preliminary data suggest that induced sputum may be a useful technique to study airway inflammation with aerosolized allergen pro~ocation,6~, 137 and to monitor effects of anti-inflammatory agents on eosinophi1 migration and activation. Table 4 summarizes 14 published studies of the application of bronchoscopy in patients with asthma, without an associated provocative stimulus. All of these studies involve a control group of normal volunteers, 13 out of the 14 studies specifically involve allergic asthmatics, but 5 studies have also investigated nonallergic asthmatics. These studies mostly involve mild asthmatics. All of the studies have excluded cigarette smokers. These studies have been performed in patients free of an acute exacerbation for at least 4 to 8 weeks, off systemic steroids for at least 8 to 12 weeks, and most often off inhaled steroids for at least 2 to 4 weeks, and with a withhold from inhaled beta-agonists for 6 to 12 hours. As can be seen from the table, there is some variation in the technique of BAL as to the location and volume of BAL used. With the exception of Bousquet et al, none of the studies performed simultaneous biopsy.2O Tables 5 and 6 summarize selected studies that

used investigative endobronchial biopsy and brushings in asthma. Several broad conclusions may be made from BAL and biopsy studies involving chronic stable asthmatics.37,74, 86 At baseline, there is no significant difference in the total number of cells in normals compared with allergic asthmatics.95,185 In general, eosinophils are not present in normals whereas asthmatics may have up to 4% to 8% eosinophils, the higher levels occurring in symptomatic allergic asthmatics.2,20, Io5, There is no significant difference in CD4,CD8, or the ratio in controls compared with allergic asthmatics.207 Endobronchial biopsies of mild asthmatics have shown increased numbers of macrophages, eosinophils, and T-lymphocytes but not ne~trophils.’~~ The numbers of mast cells are similar in asthmatic and control airways, but they have features of continual granule secretion in asthma.54Also, asthmatic airways have activated T-lymphocytes, eosinophils, and elevated numbers of high affinity IgE 89 Also, asthmatic airreceptor-bearing cells.23, ways have increased IL-4 and IL-5 mRNA and protein expression.88Asthmatic airway epithelium has increased expression of endothelin and eotaxin mRNA and protein levels.”O Studies using endobronchial brushings have shown that epithelial cells are activated and less viable in asthma.32,Io2 There is increased expression of HLA-DR and ICAM-1 in asthma.lS5Also, asthmatic airway epithelium has low levels of Cu, Zn-SOD activity that is corrected by corticosteroid therapy.” Finally, iNOS expression in the airways is maintained by IL-4 and IFN-7.” BRONCHOSCOPY FOR PROVOCATION STUDIES IN ASTHMA

A variety of models have been used to study the nature of airway inflammation in asthma. Several animal models including the guinea pig and ovalbumin have been used, but experience suggests that the specific trigger and the species studied effects the nature of the inflammatory response and animal data cannot be extended to humans. A second approach has been the study of chronic mild asthma in humans by way of BAL and endobronchial biopsies. A third approach has been the study of chronic asthma with and without anti-inflammatory therapy. Finally, other approaches include study of mild asthma with experimental provocation with a variety of

Immunohistochemistry, EM

2, lobar bronchi or carina

6 asthma 5 controls 10 asthma 9 controls

Lackie,’O* 1997

=

reverse transcriptase polymerase chain reaction; TBBx

?, subsegmental airways

? = not mentioned in study; EM = electron microscopy; RT-PCR interferon; IL = interleukin.

Lamkhioued,’lo 1997

=

transbronchial biopsy; LLL

=

left lower lobe; mm = millimeter; IFN

=

eosinophils and macrophages accumulate more in alveolar than in airway tissue in asthma

asthma airway epithelium has increased levels of eotaxin mRNA and protein

11 nocturnal asthma 10 non-nocturnal asthma

Kraft,Io61996

asthmatic airways have increased IL-4 and IL-5 mRNA and protein expression

In situ hybridization, RT-PCR

6, 2 each from right middle, lower, and upper lobes

Humbert,881996

increased expression of CD44 in asthma

Immunohistochemistry, RT-PCR, in situ hybridization Histology, EM

6, 2 each from right middle, lower, and upper lobes

12 atopic asthma 10 nonatopic asthma 10 atopic controls 12 nonatopic controls 20 asthma 17 controls

H ~ m b e r t . 81996 ~

asthmatic airways have activated T-lymphocytes and eosinophils contributing to the pathogenesis of the disease mast cell numbers are similar in asthmatic and control airways, but they have the feature of continuous granule secretion in asthma IL-5 m-RNA present in bronchial mucosa of asthmatics regulates eosinophil function in asthma endothelin is expressed in airway epithelium in asthma but not controls asthmatic airways have increased numbers of macrophages, eosinophils, and T-lymphocytes, but not neutrophils asthmatic airways have elevated numbers of high-affinity IgE receptor-bearing cells irrespective of atopic status

Summary of Findings

Histology, EM

Immunohistochemistr y

2-3, subsegmental bronchi LLL

4, fourth and fifth generation airways (also performed TBBx) ?, ?

Immunohistochemistry

?, RUL carina or subcarina

17 asthma 11 controls 16 asthma 6 controls

S ~ r i n g a l l ,1991 ’~~

P ~ s t o n . ‘1992 ~~

Immunohistochemistr y

?, subsegmental airways

In situ hybridization

Immunohistochemistry

Study Method

6, 3 subsegmental airways and 3 central airways

Number of Biopsies, Location

10 asthma 9 controls

11 asthma 9 atopic nonasthma 10 controls 11 asthma 6 controls

Patients

Hamid,8O 1991

D~ukanovic,5~ 1990

Azzawi: 1990

Study, Year

Table 5. SUMMARY OF THE RESULTS OF SELECTED STUDIES THAT USED ENDOBRONCHIAL BIOPSY THROUGH THE FLEXIBLE BRONCHOSCOPE

U

CI

0

18 controls

33 controls

16 asthma 11 controls 27 asthma 19 controls 30 CF 17 controls 13 asthma 11 controls

44 controls

Kelsen,lo21992

Erzurum," 1993

Campbell,3z1993

Guo et al,'9 1997

13 1%asthma 36 t 6% control 27 f 9% asthma 54 t 5% control ?

0.1-1 x 106 ? ?

20 subsegmental LLL

20 subsegmental LLL

? (2 mm), proximal

25 (1 mm), first and second generation bronchi

mainstem bronchi 25 (1 mm), first and second generation bronchi

asthmatic airway epithelium has low levels of Cu,Zn-SOD activity that is corrected by corticosteroid therapy iNOS expression in the airway is maintained by IL-4 and IFN-7

enzyme activity, RNA, protein

cell culture, RNA, signal transduction immunocytochemical staining ?

?

24% ciliated, 11% secretory, 29% basal, 36% other Lidocaine decreases viability low level of antioxidant enzymes in airway epithelial cells is not upregulated by hyperoxia epithelial cells are activated and less viable in asthma increased expression of HLA-DR and I-CAM in asthma decreased IL-10 expression in CF

Summary of Findings

?

cytospin, RNA

immunostaining

cell culture

RNA, enzyme activity

cell culture

Study Method

0.9 f 0.2 x 105 Asthma off steroids 2.0 f 0.7 x 105 Asthma on steroids 2.8 t 1.1 X lo5 Controls

*

?

6 (3 mm), distal tracheaproximal mainstem bronchi 13 (1mm), mainstem bronchi 16 f 2 X lo6

Viability

36 f 4%

Yield (cells)'

14 t 2 X lo6

Number of Brushes (size of brush), Location

CF = cystic fibrosis; LLL = left lower lobe; mm = millimeter; IFN = interferon; IL = interleukin. *All cell yields are expressed in total number of cells except DeRaeve, which reports as cells/bxush.

DeRae~e,4~ 1997

Bonfield,'" 1995

Vign01a,'*~1993

Patients

Study, Year

Table 6. SUMMARY OF THE RESULTS OF SELECTED STUDIES THAT USED ENDOBRONCHIAL BRUSHINGS

ROLE OF BRONCHOSCOPY IN ASTHMA RESEARCH

triggers including cold air, isocapnic hyperventilation, exercise, aerosolized hypo- or hyper-tonic saline, aerosolized antigen, or localized endobronchial or segmental allergen challenge (SAC).', 22, 31, 48, 51 The limitation of the use of bronchoscopy for simple observational studies of chronic stable asthma (without experimental provocation) is that it is generally not possible to identify important triggers, it is difficult to obtain adequate numbers of inflammatory cells for study, and it is difficult to determine the temporal relationship between the exacerbating factor and the onset and development of inflammation. For these and other reasons, many investigators have used a provocative factor.73,75 The advantage with nonallergic or nonspecific triggers is they are technically easier and are well suited to study the early phase airway response and they have the advantage of not inducing a late phase airway response. For the study of cellular and noncellular inflammatory events of the late phase, antigen challenge, however, is particularly well suited and has several distinct advantage^.^^ Antigen challenge may result in a biphasic decline in respiratory function in a sensitive, allergic asthmatic: early asthma response (EAR) that occurs within minutes and resolves by 2 hours, and a late asthmatic response (LAR) that usually occurs within 6 to 8 hours and may last for 24 hours or longer. The LAR, which occurs in 50% of adult asthmatics, is associated with increased airway reactivity to nonspecific stimuli and a cellular infiltrate in airway lavage fluid. Following antigen challenge, relationships can be constructed among the physiology, cell biology, and molecular biology of the resultant inflammation.,06 Antigen challenge is a powerful methodology for studying asthmatic airway inflamlZ5Antigen may be administered as mation.lZ4, a whole lung aerosol challenge (WLAC) or by local deposition of antigen by bronchoscopy (segmental antigen challenge or SAC).148 There are a number of limitations for the aerosol techniquez7,28: (1) The dosing of the antigen is imprecise because there is regional inhomogeneity of aerosol deposition. (2) There is lack of information regarding the distribution of the aerosol to the larger versus smaller airways. (3) Because the entire airway is challenged, it is difficult to construct dose response relationships. (4) Aerosolized antigen induces widespread bronchial obstruction; therefore, safety considerations limit the quantity of antigen that can be delivered. The

171

technique of local administration of antigen by bronchoscopy and SAC overcomes many of these limitations. SAC permits a more precise localization of antigen to small airways, a compartment that is most relevant to asthma. Higher doses can be given locally to induce more intense segmental inflammation safely, which may be more representative of natural exacerbations. This technique allows multiple segments to be used with different antigen doses for dose-response studies or the same dose for interventional or kinetic studies. Finally, this technique permits the study of novel, pharmacologic pretreatment, and their 149* 160, 162, 173, 205 effect on airway inflammati~n.~~, There, however, are a number of limitations with either WLAC / SAC. It is unclear what the relevance of the data obtained is to nonatopic asthma and clinical exacerbation of asthma most often caused by viral infections. Most of the studies have been limited to mild asthmatics and the relevance to moderate or severe asthmatics has not been studied. Because antigen is given to a localized lung segment, it is technically not possible to obtain physiologic data relevant to that particular lung segment in volunteers undergoing SAC. In addition, the optimal dose to be administered bronchoscopically is unclear and is fairly arbitrary. Most investigators have used endpoint skin titration (EPST) method and have used a fraction of the dose necessary to produce a specified size of wheal (e.g., 4 X 4 mm) to be administered by SAC. 0thers have used a screening whole lung antigen challenge to establish the dose of antigen required to produce a 20% decline in FEVl (antigen-I'D,,) during the early phase and have used various fractions of this dose for endobronchial administration (5% to 20% of A-PDZo).The problem with this approach is that the dose of antigen required to produce a predictable LAR is difficult to calculate because A-PD,, is based on EAR. There is clearly significant heterogeneity of response to SAC because of a variety of technical factors including whether the same exact segment of allergen administration is sampled by subsequent BAL and variations in local dosing. Even localized allergen challenge by SAC may be limited by systemic bronchospastic response in some patients. Most of the earlier published studies with SAC have not carefully eliminated the problem of contamination of allergen with endotoxin, which might artificially produce a neutrophilic res p o n ~ e At . ~ ~best, most SAC studies remain Text continued on page 179

v h,

CI

Baseline BAL in Ragweed, lingula 37 mL timothy x 4 grass Ag in RML, 5' dwell time, repeat BAL

Baseline BAL in Ragweed, lingula 37 mL timothy x 4 grass Ag in RML, 5' dwell, then repeat BAL

Wenzelzo2 (1988)

Wenzel'" (1990)

Ragweed, cat, alternaria

All were intubated 20mL x 5 1 control BAL 1 immediate post-Ag BAL 1 BAL at 48 or 72"

Metzger126 (1987)

Antigen

WLAC-BAL Alternaria or 2-40; ragweed SAC, immediate BAL IV TcW-Alb, SAC, BAL 20mL X 5

Technique/ Design

Fick66 (1987)

Author (year)

+

+

EPST

-

-

WLAC

Method for SAC Dosing

3

7

6

6

NV

7

ANA

11

11

15

A-A

3

3

101-99 -88 95+110

89-79 5649

63+68

Ag: 56 Control 54

62

(7.7) NS

Baseline: 7.6 48": 19.8 96": 19.6 6.7

Baseline: 8 48": 15

4.96 (8 ? 1.5)

0 25 3 1.0

2.3 NS

87 NS

1.6

P

90 40 80 82

92% NS

Mp

BAL Parameters During LAR

Total # Cells Recovery (1OE)or NA-A (mL) (x104celldml)

Patient Subgroup

Table 7. ENDOBRONCHIAL SEGMENTAL ANTIGEN CHALLENGE (SAC): SUMMARY OF PUBLISHED STUDIES

0 20 7 3.0

0.1 NS

11 NS

0.8

Eos

10 15 10 14

6%

L

Comments

By HPLC, baseline LTCn level was high in AA>ANA>NAA; with SAC, immediate t only in atopics No delayed BAL No biopsy

Baseline tryptase (T) but not histamine (H) were higher in AA; increases in T, H in atopics; non-atopics did not respond

All asthmatics had dual response Significant correlation between total dose of Ag ( p m ) and IAgl (PNU/mL) required to produce end points in skinhung; -equal total PNU were given in skin and airways Immediate visual airway change No biopsy

Post SAC, T TP in asthmatics c/w NV Immediate BAL-4 X T Tc~~-AI~; In serum proteins in BAL 2'4" without WLAC No biopsy

Baseline BAL S, Ragweed Low dose SAC, &; medium S, high S, BAL at 12 minutes, 48 hours BAL = 60mL x 2

Ragweed Skin: 2+, >10 mm to 0.02 mL 5 10 PNU/mL

D. Pterony for all NV; 1 AA; grass in 6AA

Baseline BAL RML/ling Contra-SAC BAL 24" post

RUL ant. Baseline BAL 20 mL x 6; AginRML, 10 minutes later, BAL-pooled

Ag in RML/or Ragweed, lingula; dust mite, BAL (20 x 5) timothy 18 hours later grass

Dupuis5* (1992)

Gratzioun (1992)

Georasn (1992)

x 5

Baseline BAL Ragweed Contralateral SAC BAL 10 minutes, 19 hours BAL = 20 mL

Sedgwick15' (1991)

Liu'I3 (1991)

+

-

-

-

-

+

+

+

+

c

E2 ,c El

El

EZ

10

8

5

7

6

14

6

6

10

2

1.75 1.79 1.7

45 Table continued on following page

7.7 9.3 17

13

1.91 2.34 15

16

88 86 66

26

5.2 2.0 25.6

117.9

58 NS 55 mLAg Saline+

Ag-

10

8

6.0 1.3

34 0.04 .34 .56

BAL eos, PMNs, basophils recruited to airway express more CDllb and less L-selectin than circulating cells

SAC-reduced total Tcells and decreased CDJCD, (1.58-0.99) in AA, but no change in NV (2.07-2.0) within 15 minutes. Selective retention of Tcell subtypes in EAR may be important for eos in LAR.

Higher Ag dose assoc. with higher total cells, % eos 58 0.6 1.94 2.56

0


29 12.7 89% 88%

1.8

127 14 73 mL NS 10.3 69 mL Ag 7.6

Did not identify EAR/ LAR NV No significant EAR/LAR ANA cellular response is Ag dose dependent IL-5 levels correlated with eos/ECP/ MBP/EDN

4

76%

PGF2, thromboxane LAR cellular; mediators were less elevated

EAR histamine, PGD2,

72

2

38%

28 17 21

4

9%

18

138 f 40

11 k 1 88

65

50

+

-

-

+

Calhoun28 (1993)

Montefort'** RUL ant. Seg D. Pterony or (1993) NS, grass RML+Ag; No BAL; only EBBx from saline and Ag sites @ 5-6" post SAC

Baseline BAL, Ragweed wait 24 hours WLAC, then BAL at 48" post 4 weeks SAC 20% A-PD2o BAL 48" post BAL: 60 mL x 4

NS

NS

K a t ~ ~ ~ NS or Ag in Ragweed, (1992) RML/lingula grass, dust mite BAL (20 x 5) at 19 hours

Antigen

WLAC

Technique/ Design

EPST

Author (year)

Method for SAC Dosing

c

ANA

=

8 WLAC

9 N = 8 SAC

El N

E,

NV

6

5

A-A

Patient Subgroup

=

N/A

Ag

30 10 N/A

60% 17 60

NS = 52% 43

Recovery (mL)

Total # Cells (106) or (x104/mL)

N/A

N/A

10%

70%

N/A

10%

14 5%

18 10%

30 30%

L

16

P

14

MP

65

BAL Parameters During LAR

OF PUBLISHED STUDIES (Continued)

NA-A

Table 7. ENDOBRONCHIAL SEGMENTAL ANTIGEN CHALLENGE (SAC): SUMMARY

N/A

1%

10%

39 50%

1.5

Eos

Comments

Upregulation of CAMS (E-selection, ICAM-1 but not VCAM-1) accompanied by influx of LFA-l+ leukocytes into submucosa, epithelium, including PMNs/eos+ 1 FEV,, 1PD,; influx of CD3+ T-cells @ 6"+T,,-tIL3, 4, 5, 6, GSF; VCAM-1 expression @ 24"+more selective eos, T-cells, 1 PMNs (by way of VLA-4) Biopsy - yes; EBBx from NS and Ag sites 0 5-6"post

At 48" after SAC: Increased PMA-driven SO production Increased Mp density Increased MBP, EDN

GM-CSF was 25-fold higher post SAC compared with NS GM-CSF and eos correlated in BAL No difference in ANA, AA

BAL (50 X 3) in Ragweed lingula (control) SAC in RML, with BAL (50 x 3) at 24 hours

+

+

C 5

18 27

10

V i r c h o ~ ' ~ ' Lingula+NS+ Birch/tree (1995) BAL at 18 pollen hours RLL-M-250 PNU-tBAL at 10 minutes JWL-M+250 PNU+BAL at 18 hours 25 mL aliquots x 4 = 1OOmL

Zangrillizll (1995)

27

29-50

75 105

(45)

(45)

(105 ? 30)

120

16 11

9

45 3.4

12 14

31 24

41

23 25.6

28 17

2

20 17.5

3 2

2

7.5 1.81

1.0

15

Large increase in VCAM-1 post SAC in ANA, AA Strong correlation between eos, IL-4, IL-5, sVCAM-1 Majority of increase was in patients with dual response with WLAC 37 56 Table continued on following page

0

-

-

IL-13 transcripts and proteins with SAC were higher than NS (p < 0.02) Source of IL-13 is mononuclear cells

sICAM-1 in BAL increased with SAC (p < 0.01) Small increase in SICAM-1 noted in serum Supports local release

49 12/15 59 f 20 Single/dual (AA) 11/8 (ANA) Dual Total cells: 130 ? 33 M = 29 E = 78 L = 2.8 Single Total cells = 53 -C 16 M = 21 E = 14 L = 1.36 P = 17 15 x 1 0 4 / m ~10 x lo4 5 x lo4 10 x lo4 No difference in PMN/ eos at 10 min c / w control BAL Post SAC at 18 h increase in eos/PMN/ IL-2R CD4; increased IL-5, 1L-2, IL-1, TNF, IL-6, IL-8, GM-CSF (not IL4, IFN)

27 72

110 14

Ag 48% NS

8

Control BAL Ragweed Contralateral SAC Post SAC BAL at 24 hours BAL 50 mL x 3

Fixed dose

Shaver'58 (1995)

75

16.3

NS 42%

11

NS, SAC with Ragweed, 100 or 500 timothy PNU grass BAL at 18 to 24 hours (volume N/S)

t

HuangS7 (1995)

Ragweed, grass, dust mite

NS, SAC in RML/lingula BAL (20 mL X 5) 17 to 21 hours

Takahashi'" (1994)

Antigen

Baseline BAL, Ragweed NS or SAC BAL (20 x 5) at 5 minutes, 24 hours

Technique/ Design

Gratzio~’~ RUL control, (1996) RML SAC Znd bronch @ 6 hours, 20 mL x 6 in RUL and RML

D. Pterony, grass

D. l’terony, Cruikshank3’ SAC with NS (1995) RUL, Ag RML grass BAL (20 mL x Endotoxin free 6) at 6 hours extracts

SuP7 (1995)

Author (year)

1

4

t

EPST

WLAC

Method for SAC Dosing

6

NV

ANA

N

64%

64%

5.87

7.41 56 mL AG

16

28

AG

Mp

44

19

(lo6) or (xl0VmL)

NS

Recovery (mL)

63 mL

NA-A

# Cells

Total

16%

12%

8

26

P

BAL Parameters During LAR

= 12 NS

A-A

Patient Subgroup

Table 7. ENDOBRONCHIAL SEGMENTAL ANTIGEN CHALLENGE (SAC): SUMMARY OF PUBLISHED STUDIES (Continued)

L

16%

21%

8

9

Eos

3.86

1.64

71Yo

21

Comments

Levels not detected in NV, ANA in CD,’ cells in Ag side (78-*59), no change in CD4 or CD, - reduction in monos in BAL diff can be fully accounted by loss of T-cells; No A in CD,, expression (Tcell function), but trend to t in HLADR At 6” SAC + reduction in T-cell ICAM-1 (32-+23),
K)

Lymphocyte chemoattractant activity seen in AA BAL at 6 h post SAC (mostly IL-16, MIP2-

EOS, IL-5 increase and correlate with SAC LAR

CI U U

Baseline BAL and BAL 24 hours after SAC

RUL ant. Segment control, RML SAC BAL (20 mL x 6) at 4 hours

Sur'" (1996)

Teran'16 (1996)

J a r j o ~ r ~ ~ SAC with NS and 3 (1997) doses of Ag 5 min dwell time BAL (60 mL x 2) at 5 minutes, 48 hours

NS

Hastie" (1996)

Ragweed, grass, trees, dust mite Endotoxin free extracts

Dust mite, grass

Ragweed

Ragweed

6

19

-

+

+

+

15 17 6

17

48 hours

=

70

13-24

48-84

53 85-92

Ragweed NS = 54 mL

Ag = 45 mL (11.7) 5 minutes = 75 8-12

55 45 73

38.7 58

(28.7)

73

13.8 (10.5)

55% 52% Sham

64%

.04 0-1.7

1-36 Table continued on following page

22 5-7

5-9

20 0.08

4-5

Intensity of LAR is not predictable by histamine in EAR IL-5 and BAL eos correlate with Ag dose, but GM-CSF does not Airway response in asthma is similar to ANA

Eosinophil chemotactic activity in BAL is caused by Rantes, which increased significantly with SAC (P < 0.005) Correlated with eos

34.9 .01

10.6 23 15.2 15

IL-5, Rantes, eos, EDN increased with SAC Eosinophilia correlated with IL-5 but not Rantes

Baseline ILlp levels from epithelial cells were similar in NV and allergics Levels were elevated with co-culture w/BAL cells c/w no co-culture Post-SAC levels were significant in allergies w/out co-culture

24 36 0.65

0.9

5 4 10.7

6

16 14 15

19

Antigen

2 SACS, 4 FOB, Ragweed, 6 BALs endotoxin free SAC 1 24h, day 16 SAC 2 -+ 7 day Control day 1, 9, 16: 50 mL X 3

Technique/ Design

+

EPST

WLAC 6

NV

ANA

AA-S N = 9 AA-D N = 6

A-A

NA-A

Patient Subgroup

Recovery (mL) Mp

P

BAL Parameters During LAR Total # Cells (lo6)or (xlOYmL)

L

Eos

AA-&FEV, J 46% EAR 48% LAR AA-%FEV,J 44% EAR J. 9% LAR t IL-5, GM-CSF levels in BAL or cells expressing mRNA for these in biopsies 1848h t after SAC AA-D have much more inflammatory response after SAC than AA-S No change in BAL IL-3 after SAC SAC results in prolonged eosinophilia 24h = 7 days, resolved by 16 days No biopsy

Comments

W A C = whole lung antigen challenge; SAC = segmental antigen challenge; Tc = technetium; BAL = bronchoalveolar lavage; + = yes; - = no; TG = timothy grass; RUL/RML/RLL = right upper/middle/lower lobes; EPST = end point skin titration; NV = normal volunteer; ANA = allergic nonasthmatic; A-A = allergic asthmatic; NA-A = nonallergic asthmatic; N/S = not specified; P = polymorphonuclear leukocyte; L = lymphocyte; eos = eosinophil; HPLC = high performance liquid chromatography; TP = total protein; PG = prostaglandin; ICAM = intracellular adhesion molecule; S = single or early asthma responder; D = dual phase responder; Ag = allergen; LFA = lymphocyte function-associated antigen.

Shaver'(1997)

Author (year)

Method for SAC Dosing

Table 7. ENDOBRONCHIAL SEGMENTAL ANTIGEN CHALLENGE (SAC): SUMMARY OF PUBLISHED STUDIES (Continued)

179

ROLE OF BRONCHOSCOPY IN ASTHMA RESEARCH

correlative and they do not distinguish cau(e.g., total proteins and airway permeability) sality of biologic phenomenon from a nonspe175 Also, several studduring the late phase.174, cific epiphenomenon. ies have shown an increase in IL-5 and GMTable 7 summarizes 25 published studies CSF levels in BAL and in cells expressing involving the technique of SAC. Table 8 mRNA for these cytokines from biopsies at shows the kinetics of airway inflammation 171 There seems to 18 to 48 hours after SAC.133, with SAC in a variety of allergic subjects. be no change in the IL-3 level in BAL. Also, Table 9 summarizes a variety of studies of there seems to be some selective alteration allergen bronchoprovocation showing early of cell adhesion molecules to affect selective and late phase cells and mediators and their influx of cells after SAC. Specifically, one relationship to nonspecific bronchial hyperstudy has shown a decrease in the CD, posiresponsiveness. Several broad conclusions tive cells with allergen challenge with no can be made from all these data. In general, change in CD, or CD, cells but a reduction in allergen challenge with SAC produces sigthe total mononuclear cells in the BAL.76 nificant EAR and LAR in allergic asthmatics There was no change in the CD,, expression. but not in nonasthmatic volunteer^.'^^ Allergic At 6 hours after SAC in this study, there is asthmatics have a significant increase in the a reduction in T-cell intracellular adhesion total number of cells as well as eosinophils molecule-1 (ICAM-1) and lymphocyte funcin the airway than nonallergic normals, and tion-associated antigen-1 (LFA-1). These data probably compared with allergic nonasthmamay suggest a selective interaction of intics although there have been conflicting data flammatory cell adhesion molecules with as to this.48,51, 133 In general, there is no sigmolecules present on airway epithelial or ennificant cellular influx with the early phase. dothelial cells. EAR, however, is marked by increases in histamine, tryptase, prostaglandin D,, prostaglandin Flr thromboxane, and leuk~trienes.~~,BRONCHOSCOPYTOASSESS 113, 130, Studies have shown cellular influx RESPONSE TO ANTIby 6 to 8 hours with an initial increase in INFLAMMATORY THERAPY neutrophils followed by an increase in eosinophils from 24 to 48 Recent studies A variety of studies have been performed suggest that the eosinophilic inflammation in in asthmatics of varying severity to assess BAL continues to be present at day 9 but anti-inflammatory effects of therapy by bronseems to resolve by day 16.159Other studies choscopy with lavage or biopsy.6 Table 10 using SAC have shown that during EAR there summarizes 10 recent studies assessing the is a reduction in the total number of T-cells anti-inflammatory effect of corticosteroids by and a reduced CDJCD, ratio in allergic asththe use of investigative bronchoscopy. Most matics but not in normal volunteers sugof these studies have been performed using gesting a selective retention of T-cell subtypes small numbers of patients, usually with mildin the EAR, which may be important for subto-moderate asthma treated with moderatesequent elaboration of selective cytokines and to-high doses of inhaled corticosteroids for a eosinophilia during LAR.77 duration varying between 2 weeks and 5 to Other studies using the SAC model have 10 years. These studies have typically shown shown increases in noncellular components more significant improvement in parameters Table 8. KINETICS OF DEVELOPMENT OF INFLAMMATION AFTER SEGMENTAL ANTIGEN CHALLENGE IN ALLERGIC ASTHMATICS

Type Total Cells (Millions) PMN (Millions) EOS (Millions)

0 hours

8 hours

24 hours

48 hours

P (ANOVA)

NS <0.001 NS <0.001 NS <0.004

Nv

19.7

15.0

13.6

23.7

AA

12.3

28.2

29.2

48.2*

Nv

0.21

1.4

0.6

1.8

AA

0.29

7.9,

3.1

Nv

0.00

AA

0.10

0.001 5.9

1.9 0.04 19.3*

0.06 6.3

“R0.05 for indicated time versus each other time point; NV = normal volunteer (normal type); AA = allergic asthmatic (bold type) From Calhoun WJ, Friedenheim RE, Vuchinich T, et al: Kinetics of development of inflammation after segmental antigen challenge (SAC) in allergic asthmatics. Am J Respir Crit Care Med 151:A703, 1995; with permission.

180

KAVURU et a1

Table 9. SUBSTANCES ELEVATED IN THE AIRWAYS OF ALLERGIC ASTHMATICS, EITHER UNCHALLENGED OR AFTER ALLERGEN CHALLENGE

Substance Cells Mast Cell N or % Mast Cell Degranulation Basophils Eosinophils Neutrophils Epithelials Monocyte/macrophages CD8+ T cells CD4+ T cells Mediators Histamine Tryptase PGD2 9-alpha, 11-beta PGF2 LTC4, D4, or E4 PGE2 PGF2 alpha Thromboxane Kinins, kallikrein Cytokines (protein or mRNA) TNF IL-1 beta IL-2 IL-3 IL-4 IL-5 IL-6 GM-CSF Other substances ECP EPO Endothelin MBP EDN CLC protein Albumin, urea Hyaluronin LYSO-PAF DNA a

Unchallenged Asthmatics"

Challenged Allergics or Asthmaticsb

BAL or sputum

BAL-Acute

1

f

t

N

T

N N

TtN

t

N, t ( C H )

?t

tt t T t 't

t t t tT

t t

t t t

BAL-Late

t 1

ttt ttt ttt

tTt tT

T t t T t

t t t t t ?t t Tt

tt tt tt t

tTt

tT

t

f t

f f

tt

tt

1'

t t t t

tt

T T

tT

Tt

t t

ttt ttt

= compared with controls without asthma; b = compared to pre-challenge levels. = increased, = decreased; N = normal; no arrow indicates that the levels are either unchanged or have not been determined; MBP = major basic protein; ECP = eosinophil cationic protein; EDN = eosinophil-derived neurotoxin; TNF = tumor necrosis factor.

t

1

Modified from Bochner BS, Undem BJ, Lichtenstein LM Immunological apsects of allergic asthma. Ann Rev Immunol 12307, 1994; and Smith DL, DeShazo RD: Bronchoalveolar lavage in asthma: an update and perspective. Am Rev Respir Dis 148523,1993.

of inflammation in endobronchialbiopsy than on a corresponding BAL cellular pr~file.'~, 52* 134, 179, 203 Most studies have reported a marked post-treatment reduction in T-lymphocytes, eosinophils, and mast cells in the lamina propria. Poststeroid therapy biopsies have also shown a decrease in cells expressing mRNA for IL-4, IL-5, and GM-CSF along with an increase in IFN-y cells.12The reports on the effect of inhaled steroids on thickness of the epithelium and basement membrane have been inconclusive with at least one study

showing no improvement despite therapy for 10 years.115Only one study has evaluated severe steroid-dependent asthmatics by the use of BAL and biopsyzo3Wenzel et a1 studied 14 patients being treated with prednisone (mean dose 30 mg/d), in addition to inhaled steroids ranging between 800 and 4000 mcg/day for a duration of more than 5 years.2o3BAL from these patients continued to show elevated levels of LTB4, LTE4, thromboxane, and histamine. Although there was no significant difference in the total number of BAL cells, the

60 mL x 3 (lingula); no change in any BAL parameters

8 weeks

4 months

BDP 500 mcg BID

BDP 500 mcg BID vs. placebo

N

N = 25 w/mild asthma (PC,, = 0.11 mg/mL) FEV, increased 11% in both groups (no difference)

Randomized, placebo-controlled

Randomized, placebo-controlled

Sousa’@(1993)

(1994)

14

60 mL X 3 (RML) Decrease in total cells (from 187 to 163 x 103/mL) as well as eos, epithelial, and mast cells Increase in lymphs Not performed

3 months

BDP 1000 mcg BID

= 20 asthmatics (variable, mild)

N

Uncontrolled, pre-, post-

Duddridge5’ (1993)

=

Not performed

3 months

BUD 400 pg BID

= 6 mild-moderate asthma 0.69 mg/mL)

Uncontrolled pre-, post-

N

Not performed

10 years

BUD 400-1600 mcg/d

BAL Findings

Duration

Drug

Burke” (1992)

Lundgren115(1988)

6 severe asthma 6 healthy controls

Subjects = =

Design

N N

Author (year)

Table 10. BRONCHOSCOPY STUDIES TO ASSESS ANTI-INFLAMMATORY THERAPY WITH CORTICOSTEROIDS

Asthmatic airway epithelial cells express more GM-CSF than normals Significant reduction only in BDP group ( P = 0.014) Mast cells, eos in mucosa were reduced in BDP ( P = 0.05) GM-CSF expression in epithelium was reduced from 25% to 12% ( P = 0.008) in BDP only; BM thickness decreased from 29.7 to 19.8 p,m ( P = 0.04) No change in CD,/CD8 Table continued on following page

( P < 0.05) No change in thickness of epithelium No change in BM thickness (7 to 5.8 pm) 2 to 4 endobronchial biopsies Significant reduction in infiltrating T-cells, CD,, RO+ cells, RFDl+ dendritic cells, and HLA-DR expression in the inflammatory infiltrate after BUD therapy No biopsy

1 inflammatory cells

BrushlBiopsy Findings

60 mL X 4 RML No significant difference in total cells, mild asthma had more eos, severe asthma had more polys Elevated levels of LT-B,, LT-E,, thromboxane, histamine

Post FP, marked reduction in T-lymphs, T-cell subsets, activated T-cells (CD,' cells) and EG,' eosinophils in the lamina propria Decrease in CD,+-T-cells, activated EG2' eos, mucosal-type mast cells; Decrease in cells expressing mRNA for IL-4, IL-5; increase in IFN-y cells In prednisolone group: decrease in submucosal mast cells, eos, CD,+-Tcells No change in control group Eos and mast cells in lamina propria were decreased in FP ( P < 0.01) BM thickness decreased only in FP group (P < 0.05) 4 to 6 biopsies RLL Inflammatory infiltrate present with excess polys, few eos No difference between EBBx and TBBX

BrushlBiopsy Findings

BDP = beclomethasone dipropionate; BUD = budesonide; FP = fluticasone propionate; bid = twice per day; RUL, RML, RLL = right upper, middle, and lower lobes; BM = basement membrane; PC,, = provocative concentration of histamine or methacholine to reduce FEV, by 20%; ECP = eosinophil cationic protein; EBBx, TBBX = endobronchial and transbronchial biopsy; LT = leukotriene; IL = interleukin; IFN = interferon.

>5 years

Mean prednisone dose = 39 mg Inhaled steroids 8004000 mcg/d

N = 14 Severe, steroid-dependent asthma tics

Prospective, observational with historical controls

WenzeFo3(1997)

50mL X 3 No change in cellularity FP group: decrease in cells expressing ICAM-1, MAC-1; tryptase but not ECP decreased 6 weeks

FP 250 mcg BID

N = 20 Mild-moderate symptomatic asthmatics, no inhaled steroid at baseline

Randomized, placebo-controlled

Olivieri'" (1997)

Not performed

2OmL X 6,RUL/LUL No change in any cellular parameters with therapy or between groups

2 weeks

60mL X 3RML Post FP, decrease in tryptase, lymphocytes No change in proportion of eos in BAL

BAL Findings

6 weeks

Randomized, placebo-controlled

D~ukanovic~~ (1997)

P.O. prednisolone (0.6 mg/kg/d)

3 months

Duration

Prednisolone: 20 mg/d x 2 weeks 10 mg/d X 4 weeks

Randomized, placebo-controlled

Bentley', (1996)

Drug

N = 27 Mild-moderate atopic asthmatics, symptomatic, no inhaled steroid

26 healthy controls 20 mild-moderate asthma (PD,, = .22 mg/ mL) (on PRN salbutamol, <400 mcg/d BDP) N = 18 moderate-severe asthma (PC, = 0.60 mg/ mL) = =

Single blind, placebocontrolled

Boothrg(1995) N N

Subjects

Design

Author (year)

Table 10. BRONCHOSCOPY STUDIES TO ASSESS ANTI-INFLAMMATORY THERAPY WITH CORTICOSTEROIDS (Continued)

ROLE OF BRONCHOSCOPY IN ASTHMA RESEARCH

more severe asthmatics had greater numbers of neutrophils, whereas the milder asthmatics had more eosinophils. This same pattern of cellularity was also seen on biopsy specimens. There was no significant difference between endobronchial biopsy and transbronchial biopsy in this group. Several investigators have used a variety of other anti-inflammatory agents to assess their effectiveness using the SAC model. Agents studied have included nedocromil ~ileuton,9~ zafir1ukast;O combination of zafirlukast and loratadine,15*and a nonselective nitric oxide synthase inhibitor (L-NAME).’” In summary, a variety of studies suggest that bronchoscopy is a valid approach to assess anti-inflammatory effects of various agents, particularly inhaled corticosteroids. These agents bring about a variety of antiinflammatory effects in the submucosal infiltrate. There are conflicting data as to whether long-term therapy with inhaled corticosteroids reduces basement membrane thickening and substantially affect airway remodeling. Further data are required in this area. Beyond inhaled corticosteroids, there are limited data using research FOB to support anti-inflammatory effect of other agents. SUMMARY

Over the past 15 years, much has been learned about the presence of airway inflammation in asthma through the use of investigative bronchoscopy. It has become quite clear that inflammation is present even in mild asthma. In addition to the eosinophils, T-lymphocytes and a variety of cytokines have been identified to play a prominent role in asthmatic inflammation. The concept of delayed asthmatic response after allergen exposure and its relationship to cellular inflammation and airway hyper-reactivity has become more clearly established. Our understanding of asthmatic airway inflammation, however, is i n ~ o m p l e t e . ~ ~ As interesting as the database has been so far, investigative FB has not defined a unique profile for patients with asthma. Specifically, lavage or endobronchial biopsy has not identified parameters that help in the diagnosis, assessment of disease severity, prognosis, or likelihood to respond to specific therapies. Also, the exact relationship between parameters in lavage compared with mucosal biopsy and how these are related to airway hyper-

183

reactivity and the clinical syndrome of asthma remains poorly understood. In this regard, it must be confessed that currently FB with lavage and biopsy in asthmatics needs to be considered as a research tool for specimen retrieval to help characterize and express inflammation. Although these techniques have contributed immensely to our understanding of asthma pathogenesis, presently these techniques do not have any practical role or clinical usefulness. References 1. Aalbers R, Kauffman HF, Vrugt B, et al: Allergeninduced recruitment of inflammatory cells in lavage 3 and 24 h after challenge in allergic asthmatic lungs. Chest 103:1178-1184, 1993 2. Adelroth E, Rosenhall L, Johansson SA, et al: Inflammatory cells and eosinophilic activity in asthmatics investigated by bronchoalveolar lavage. Am Rev Respir Dis 142:91-99, 1990 3. Alam R, York J, Boyars M, et al: Increased MCP-1, RANTES, and MIP-1 in bronchoalveolar lavage fluid of allergic asthmatic patients. Am J Respir Crit Care Med 153:1398-1404,1996 4. Azzawi M, Bradley B, Jeffery PK, et al: Identification of activated T lymphocytes and eosinophils in bronchial biopsies in stable atopic asthma. Am Rev Respir Dis 1421407-1413, 1990 5. BAL Cooperative Group Steering Committee: Bronchoalveolar lavage constituents in healthy individuals, idiopathic pulmonary fibrosis, and selected comparison groups. Am Rev Respir Dis 14151695202, 1990 6. Bames PJ: Anti-inflammatory therapy for asthma. Annu Rev Med 44:229-242, 1993 7. Baughman R, Strohofer S, Kim CK Variation of differential cell counts of bronchoalveolar lavage fluid. Arch Pathol Lab Med 110:341-343, 1986 8. Bavbek S, Kalaycioglu 0, Beder S, et al: Endoscopic scoring system in patients with allergic rhinitis. J Investig Allergol Clin Immunol 7175-178, 1997 9. Beasley R, Roche WR, Roberts JA, et al: Cellular events in the bronchi in mild asthma and after bronchial provocation. Am Rev Respir Dis 139:80&817, 1989 10. Bell DY, Haseman JA, Spock A, et al: Plasma proteins of the bronchoalveolar surface of the lungs of smokers and non-smokers. Am Rev Respir Dis 12472-79, 1981 11. Bentley AM, Durham SR, Robinson DS, et al: Expression of endothelial and leukocyte adhesion molecules intercellular adhesion molecule-l, E-selectin, and vascular cell adhesion molecule-1 in the bronchial mucosa in steady-state and allergen-induced asthma. J Allergy Clin Immunol92:857-868, 1993 12. Bentley AM, Hamid Q, Robinson DS, et al: Prednisolone treatment in asthma: Reduction in the numbers of eosinophils, T cells, Tryptase-only positive mast cells, and modulation of IL-4, IL-5, and interferon-gamma cytokine gene expression within the bronchial mucosa. Am J Respir Crit Care Med 153~551-556,1996 13. Bernstein IL, Boushey HA, Chemiack RM, et al:

184

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NHLBI workshop summaries: Summary and recommendations of a workshop on the investigative use of fiberoptic bronchoscopy and bronchoalveolar lavage in asthmatics. Am Rev Respir Dis 132:180182, 1985 14. Bleeker ER, McFadden ER, Boushey HA, et al: Investigative use of bronchoscopy, lavage and bronchial biopsies in asthma and other airways diseases. Eur Respir J 5:115-121, 1992 15. Bochner BS, Undem BJ, Lichtenstein L M Immunological aspects of allergic asthma. Annu Rev Immuno1 12:295-335,1994 16. Bonfield TL, Konstan MW, Burfeind P, et a1 Normal bronchial epithelial cells constitutively produce the anti-inflammatory cytokine interleukin-10, which is downregulated in cystic fibrosis. Am J Respir Cell Mol Biol 13:257-261, 1995 17. Booth H, Richmond I, Ward C, et al: Effect of high dose inhaled fluticasone propionate on airway inflammation in asthma. Am J Respir Crit Care Med 15245-52, 1995 18. Borish L, Aarons A, Rumbyrt J, et a1 Interleukin10 regulation in normal subjects and patients with asthma. J Allergy Clin Immunol971288-1296, 1996 19. Borish L, Rosenwasser L Thl/Th2 Lymphocytes: Doubt some more. J Allergy Clin Immunol 99:161164, 1997 20. Bousquet J, Chanez P, Lacoste JY, et al: Eosinophilic inflammation in asthma. N Engl J Med 323:10331039, 1990 21. Braun J, Mehnert A, Dalhoff K, et al: Different BALF protein composition in normal children and adults. Respiration 64350-357, 1997 22. Broide DH, Firestein GS: Endobronchial allergen challenge in asthma: Demonstration of cellular source of granulocyte macrophage colony-stimulating factor by in situ hybridization. J Clin Invest 88:104&1053, 1991 23. Burastero SE, Borgonovo B, Gaffi D, et al: The repertoire of T-lymphocytes recovered by bronchoalveolar lavage from healthy nonsmokers. Eur Respir J 9:319-327, 1996 24. Burke C, Power CK, Norris A, et al: Lung function and immunopathological changes after inhaled corticosteroid therapy in asthma. Eur Respir J 5:73-79, 1992 25. Bums DM, Shure D, Francoz R, et al: The physiologic consequences of saline lobar lavage in healthy human adults. Am Rev Respir Dis 127695-701,1983 26. Calhoun WJ, Dick EC, Schwartz LB, et al: A common cold virus, rhinovirus 16, potentiates airway inflammation after segmental antigen bronchoprovocation in allergic subjects. J Clin Invest 94:22002208,1994 27. Calhoun WJ, Friedenheim RE, Vuchinich T, et a1 Kinetics of development of inflammation after segmental antigen challenge (SAC) in allergic asthmatics. Am J Respir Crit Care Med 151:A703, 1995 28. Calhoun WJ, Jarjour NN, Gleich GJ, et al: Increased airway inflammation with segmental versus aerosol antigen challenge. Am Rev Respir Dis 14714651471, 1993 29. Calhoun WJ, Jarjour NN, Gleich GJ, et a1 Effect of nedocromil sodium pretreatment on the immediate and late responses of the airway to segmental antigen challenge. J Allergy Clin Immunol 9854650, 1996 30. Calhoun WJ, Lavins BJ, Minkwitz MC, et al: Effect of zafirlukast (accolate) on cellular mediators of in-

flammation. Bronchoalveolar lavage fluid findings after segmental antigen challenge. Am J Respir Crit Care Med 1571381-1389, 1998 31. Calhoun WJ, Reed HE, Moest DR, et al: Enhanced superoxide production by alveolar macrophages and air-space cells, airway inflammation, and alveolar macrophage density changes after segmental antigen bronchoprovocation in allergic subjects. Am Rev Respir Dis 145:317-325, 1992 32. Campbell AM, Chanez P, Vignola AM, et al: Functional characteristics of bronchial epithelium obtained by brushing from asthmatic and normal subjects. Am Rev Respir Dis 147529-534, 1993 33. Carre PH, Laviolette M, Belanger J, et al: Technical variations of bronchoalveolar lavage (BAL): Influence of atelectasis and the lung region lavaged. Lung 163~117-125,1985 34. Chetta A, Foresi A, Del Donno M, et al: Airways remodeling is a distinctive feature of asthma and is related to severity of disease. Chest 111:852-857, 1997 35. Chetta A, Foresi A, Del Donno M, et al: Bronchial responsiveness to distilled water and methacholine and its relationship to inflammation and remodeling of the airways in asthma. Am J Respir Crit Care Med 153:910-917, 1996 36. Chilton FH, Averill FJ, Hubbard WC, et al: Antigeninduced generation of lyso-phospholipids in human airways. J Exp Med 183:2235-2245, 1996 37. Collins DS, Dupuis R, Gleich GJ, et a1 Immunoglobulin E-mediated increase in vascular permeability correlates with eosinophilic inflammation. Am Rev Respir Dis 147677483, 1993 38. Crimi E, Chiaramondia M, Milanese M, et al: Increased numbers of mast cells in bronchial mucosa after the late-phase asthmatic response to allergen. Am Rev Respir Dis 144:1282-1286,1991 39. Cruikshank WW, Long A, Tarpy RE, et al: Early identification of interleukin-16 (lymphocyte chemoattractant factor) and macrophage inflammatory protein la (MIPla) in bronchoalveolar lavage fluid of antigen-challenged asthmatics. Am J Respir Cell Mol Biol 13738-747, 1997 40. Dahl R, Pedersen B, Venge P: Bronchoalveolar lavage studies. Eur Respir Rev 1:272-275, 1991 41. Daniele RP, Altose MD, Rowlands DT Jr: Immunocompetent cells from the lower respiratory tract of normal human lungs. J Clin Invest 56986-995,1975 42. Daniele RP, Elias JA, Epstein PE, et a1 Bronchoalveolar lavage: Role in the pathogenesis, diagnosis, and management of interstitial lung disease. Ann Intern Med 102:93-108,1985 43. Dauber JH, Rossman MD, Daniele RP: Bronchoalveolar cell populations in acute sarcoidosis. Observations in smoking and non-smoking patients. J Lab Clin Med 94862-871, 1979 44. Davis GS, Giancola MS, Costanza MC, et al: Analyses of sequential bronchoalveolar lavage samples from healthy human volunteers. Am Rev Respir Dis 126:611-616, 1982 45. de Blasio F, Daughton DM, Thompson AB, et al: General vs local anesthesia; effect on bronchoalveolar lavage findings. Chest 104:1032-1037,1993 46. de Gouw HWFM, Hendriks J, Woltman AM, et al: Exhaled nitric oxide (NO) is reduced shortly after bronchoconstriction to direct and indirect stimuli in asthma. Am J Repir Crit Care Med 158:315-319,1998 47. de la Fuente PT, Romagnoli M, Godard P, et al: Safety of inducing sputum in patients with asthma

ROLE OF BRONCHOSCOPY IN ASTHMA RESEARCH

48.

49.

50.

51.

52.

53.

54.

55.

56.

57.

58.

59.

60.

61.

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63.

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