Distal lung inflammation in asthma

Review article Supported by a grant from AstraZeneca LP

Distal lung inflammation in asthma E. Rand Sutherland, MD and Richard J. Martin, MD

Objective: To review the role of distal lung inflammation in asthma. Data Sources and Study Selection: Selected peer-reviewed research publications retrieved from MEDLINE search. Search was restricted to English-language publications only. Included articles were selected for their relevance to pathophysiology, diagnosis, and treatment. Bibliographies of selected papers served as an additional source of considered publications. Results: Inflammation in the small airways and alveolar tissue plays an important role in the clinical manifestations of asthma. Diagnostic modalities such as transbronchial biopsy and evolving radiologic techniques such as high-resolution computed tomography are improving our ability to evaluate this portion of the lung. New ultra-fine particle size metered-dose inhalers and oral agents such as cysteinyl leukotriene receptor antagonists offer new opportunities for treating small airways inflammation and need to be fully evaluated for their ability to target and treat distal lung inflammation. Conclusions: Distal lung inflammation is an important component of airway inflammation in asthma. New modalities for evaluating distal airway inflammation and for targeting the distal lung with inhaled and systemic drugs are rapidly expanding our knowledge of the clinical importance of distal lung inflammation and may ultimately be of critical importance in asthma therapy. Ann Allergy Asthma Immunol 2002;89:119–124.

INTRODUCTION Airway inflammation is a sine qua non of asthma. Much of our understanding of airway inflammation in asthma has been derived from evaluation of the large airways by measures such as induced sputum and proximal airway endobronchial biopsies. In recent years, however, physiologic and transbronchial biopsy studies of the distal lung have allowed researchers to understand more fully the role of small airway and alveolar tissue inflammation in asthma. The small airways are generally defined as those distal to the seventh or eighth ramification of the tracheobronchial tree, with an inner lumenal diameter of less than 2 mm. In addition to inflammation of the airways themselves, inflammation in the alveolar tissue is also a significant finding in asthma, and we will use the terms “small airways” and “distal lung” to refer to the combination of the terminal airways and adjacent alveolar tissue. This article will discuss the importance of the distal lung with regard to asthma pathophysiology, will summarize the contribution of distal lung inflammation to the clinical manifestations of asthma, and will discuss the evolving role of new inhaled and oral pharmaceutical preparations in the treatment of distal lung inflammation. Physiology of the Distal Lung in Asthma In 1990, Wagner et al1 used the technique of peripheral airways resistance measurement via a wedged bronchoscope Department of Medicine, National Jewish Medical and Research Center and University of Colorado Health Sciences Center, Denver, Colorado Received for publication January 5, 2002. Accepted for publication in revised form April 9, 2002.

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to evaluate distal lung physiology in normal subjects and subjects with asymptomatic, mild asthma. They wedged a bronchoscope in a subsegmental right upper lobe bronchus and progressively increased airflow through one port of the bronchoscope while measuring resulting airway pressure changes through a second port. Using the physiologic relationship airway resistance ⫽ airway pressure/rate of airflow, they were able to evaluate peripheral airway resistance. In asthmatic subjects, increases in airflow resulted in increases in peripheral airways resistance. This finding was not seen in healthy control subjects, however, despite the fact that at baseline the two groups had similar lung function (Fig 1). The authors were also able to demonstrate a correlation between airway responsiveness and increased peripheral airways resistance. These observations demonstrated the importance of increased peripheral airways resistance in even the mildest of asthmatic subjects.1 In the normal lung the parenchyma is closely attached, or coupled, to the airways. This leads to a stereotypical relationship between lung volumes and airway resistance such that airways resistance declines with increasing lung volumes, due in large part to tethering of the airways by the parenchyma. In nocturnal asthma, however, distal lung inflammation (described below) alters the normal relationship between the airways and lung parenchyma, resulting in uncoupling of the two and an alteration in the relationship between lung volume and airway resistance. In 2000, Irvin et al2 published the results of a study of the interdependence of airways and lung parenchyma in the patient with nocturnal asthma. They studied five subjects with

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Figure 1. Relationship between pressure (PB, cm H2O) and flow (V) in normal subjects (f) and asthmatic subjects (䡺). Reprinted from Reference 1.

nocturnal asthma using whole-body plethysmography during both sleep and wakefulness to evaluate the degree of interdependence of the parenchyma and airways as evaluated with volume-resistance relationships. They were able to demonstrate that lower pulmonary resistance (ie, resistance below the glottis, Rlp) increased during sleep in subjects with nocturnal asthma, from 16.2 ⫾ 2.8 to 29.8 ⫾ 9.6 cm H2O/L/ second (P ⬍ 0.001). When continuous negative pressure (CNP) was applied to the thorax using a poncho cuirass to produce a thoracic gas volume (TGV) which approximated the subjects’ awake TGV, airways resistance failed to fall significantly, with an Rlp at TGV of 29.8 ⫾ 9.6 cm H2O/L/ second versus an Rlp at TGV ⫹0.8 L of 26.6 ⫾ 7.4 cm H2O/L/second (Fig 2). In two subjects, airways resistance increased with this procedure. Over the course of the night, the amount of CNP required to increase TGV progressively increased. Respiratory system compliance was derived from the relationship between changes in CNP and resultant changes in thoracic gas volume. From early sleep to late sleep, respiratory system compliance fell from 0.079 ⫾ 0.02 cm H2O/L to 0.035 ⫾ 0.002 cm H2O/L, indicating that nocturnal asthma may be associated with up to a 50% fall in respiratory system compliance.2 Differences Between Large and Small Airway Inflammation in Asthma Histologic evaluation of autopsy specimens from severe asthmatic patients has allowed airway inflammation to be evaluated simultaneously in both the large and small airways. Carroll et al3 reported that lymphocytes and eosinophils were uniformly distributed not just throughout the proximal but also the distal lung in autopsy specimens from patients with both fatal and nonfatal asthma.3 In 1998, Haley et al4 reported that the large airways (outer perimeter ⬎3.0 mm) of patients with asthma had a greater density of CD45-positive lymphocytes (P ⬍ 0.05) and eosinophils (P ⬍ 0.001) in the region of the subbasement membrane and airway smooth muscle than

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Figure 2. The effect of sleep on volume-resistance relationships in nocturnal asthma. During wakefulness (E) the volume-resistance relationship is intact as increasing lung volume produces a fall in lower airway resistance (Rla). During sleep (F) Rla increases. The lung is inflated with continuous negative pressure applied to the thorax, Rla does not fall appropriately. This indicates that “uncoupling” of the lung parenchyma and airways has occurred, indicating loss of the normal volume-resistance relationship. Data points are mean ⫾ SEM for the five subjects. Reprinted from Reference 2.

did the same region of the small airways. By contrast, in the small airways, there was a greater density of CD45-positive lymphocytes (P ⬍ 0.01) and eosinophils (P ⬍ 0.01) in the outer (the area between the airway smooth muscle and alveolar attachments) as opposed to the inner airway region.4 The regional variations in inflammatory cell distribution seen within asthmatic airways by Haley et al4 were not observed in autopsy specimens from patients with cystic fibrosis and may represent an important difference between the inflammatory phenotype in severe asthma versus other severe obstructive airways diseases. Pathology of Small Airways Inflammation in Nocturnal Asthma In 1996, Kraft et al5 evaluated alveolar tissue inflammation in patients with chronic, stable asthma. They studied 21 patients, 11 of whom had nocturnal asthma and 10 of whom had nonnocturnal asthma. Each subject underwent bronchoscopy with endobronchial and transbronchial biopsy at both 4 AM and 4 PM, and morphometric analysis was used to determine the number and type of inflammatory cells per unit volume. The authors showed that in alveolar tissue biopsies obtained at 4 AM (Fig 3), there were significant increases in the number of eosinophils per unit volume in nocturnal asthmatic patients when compared with nonnocturnal asthmatic patients (median 40.2 ⫻ 103, and interquartile range (IQ) 26.4 to 57.1 ⫻ 103 versus median 15.7 ⫻ 103 and IQ 2.1 to 35.2 ⫻ 103, P ⫽ 0.05 for the comparison). Similar differences could not be demonstrated in proximal airway endobronchial biopsies. Further, in subjects with nocturnal asthma, there was a circadian variation in the number of eosinophils seen in

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necrosis factor, interleukin (IL)-4, IL-5, and IL-13 are produced by T-lymphocytes and facilitate eosinophil migration into tissues through vascular endothelium. In 1999, Kraft et al7 studied whether lymphocytes were responsible for recruitment of eosinophils into alveolar tissue in patients with nocturnal asthma. They demonstrated that at 4 AM nocturnal asthmatic patients had significantly greater numbers of CD4positive lymphocytes in alveolar tissue than did nonnocturnal asthmatic patients (median 9.8 per mm2, and IQ 5.6 to 30.8 vs median 1.5 per mm2, and IQ 0 to 6.3, P ⫽ 0.04 for the comparison). Alveolar CD4-positive cells correlated positively with the number of EG2⫹ eosinophils (r ⫽ 0.66, P ⫽ 0.01) and inversely with forced expiratory volume in 1 second (r ⫽ – 0.68, P ⫽ 0.0018) at 4 AM.7

Figure 3. Transbronchial biopsies performed at 4:00 AM showing distal lung tissue from a subject with nocturnal asthma (panel A) and a subject with nonnocturnal asthma (panel B). Stain, hematoxylin-eosin and azure. Arrows indicate eosinophils. A ⫽ alveolar space, V ⫽ vessel. Reprinted from Reference 5.

alveolar tissue, with a significantly greater number of eosinophils present at 4 AM (41.4 ⫾ 8.6 ⫻ 103 vs 12.9 ⫾ 4.3 ⫻ 103, P ⫽ 0.005). The number of macrophages was also greater in nocturnal asthmatic alveolar tissue at 4 AM than at 4 PM. No circadian differences in the number of large airway inflammatory cells were demonstrated. In nonnocturnal asthmatic patients, circadian differences in alveolar inflammatory cell counts could not be demonstrated. Increases in alveolar eosinophils in nocturnal asthmatic patients correlated with nocturnal decrements in forced expiratory volume in 1 second (r ⫽ – 0.54, P ⫽ 0.03); this relationship was not seen in large airway tissue. The authors concluded that alveolar tissue inflammation was an important component of the inflammatory response in asthmatic patients, and that circadian variations in alveolar tissue inflammation may play an important role in the pathogenesis of nocturnal asthma.5 T-lymphocytes are also thought to play a central role in asthma pathogenesis.6 Inflammatory cytokines such as tumor

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Radiographic Evaluation of the Small Airways in Asthma Although conventional chest radiographs and computed tomography have historically been limited in their ability to detect changes in the small airways, high resolution computed tomography (HRCT) can adequately image airways with a lumenal diameter as small as 1.5 to 2.0 mm, and a wall thickness as small as 0.25 mm.8 This has facilitated a number of studies of the radiographic appearance of the small airways in asthma. In 1996, Okazawa et al9 used quantitative image analysis demonstrated with HRCT to demonstrate significant bronchial wall thickening in the small airways (⬍ 6 mm diameter) of asthmatic patients, resulting in an increased bronchial wall area in the small airways of asthmatic patients when compared with normal controls. The authors also used this technique to demonstrate that methacholine-induced bronchoconstriction occurred primarily in airways 2 to 4 mm in diameter in both normal and asthmatic subjects. This bronchoconstriction was associated with a decrease in total airway wall area in normal subjects but not in asthmatic patients, most likely as a result of the asthmatic bronchial wall thickening noted above.9 In 1998, Goldin et al10 reported that methacholine induced bronchoconstriction in up to 95% of airways with a diameter between 1.6 and 2.5 mm. Obstruction and narrowing of the small airways causes trapping of gas and hyperinflation of lung units, which can also be evaluated with HRCT by evaluating differences in lung attenuation. By performing HRCT at different lung volumes in the same subject, lung attenuation curves may be generated. These curves further define the distribution of lung attenuation in a lung segment of interest and, when obtained at different points in time, allow the relationship between airway structure and function with regard to ventilation to be evaluated more fully by assessing alterations in gas trapping over time.10 Inhaled Corticosteroids and Distal Lung Inflammation In 1987, an international agreement known as the Montreal Protocol11 mandated the gradual elimination of substances that deplete the ozone layer, among them chlorofluorocarbons (CFCs), which traditionally have been used as propel-

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lants in pressurized metered-dose inhalers (MDIs).12 This mandate has led to the development of alternative propellants for MDIs, among them the hydrofluoroalkanes (HFA). Additionally, dry powder inhalers (which deliver medications by means of the patient’s inspiratory effort) have become more prominent in the therapeutic armamentarium in the United States. Inhaled corticosteroids are recommended as the first-line controller agent for the treatment of persistent asthma.13 Five different inhaled corticosteroids (beclomethasone dipropionate [BDP], budesonide, flunisolide, fluticasone, and triamcinolone) are currently available in the United States, and some inhaled corticosteroids are available in a variety of formulations, ranging from CFC-MDI to dry powder inhalers. The particle size generated by these devices is critical to determining where in the lung the drug is deposited. Particle size is inversely related to the depth of penetration of the particle into the bronchial tree, and the efficacy of inhaled corticosteroids may be greatest when they are deposited in the smaller airways.14 One limitation of CFC and dry-powder drug deliver devices has been that, because in part of the size of the inhalable particle they generate, only 5 to 30% of the drug is delivered to the lungs15; the remainder of the aerosolized drug is deposited in the oropharynx, where it may be unpredictably or incompletely absorbed. Alterations in the propellant used to pressurize MDIs have also allowed changes to be made in the formulation of the drug. In the case of BDP, the use of HFA as a propellant has allowed the drug to be reformulated as a solution rather than as a suspension. The combination of the HFA propellant and BDP in solution results in a particle size that is smaller than that produced by the traditional CFC-BDP (1.1 ␮m vs 3.5 to 4.0 ␮m mass median aerodynamic diameter).16 Clinical trials of this preparation have suggested that there is improved drug delivery to the distal lung and equivalent clinical efficacy at reduced doses with HFA-BDP versus CFC-BDP.17 In 1998, Leach et al18 demonstrated that the smaller particle size was associated with improved delivery of drug to the airways. Using technetium-99m-radioloabled beclomethasone delivered via CFC- or HFA-powered MDI, the authors demonstrated a significant increase in airway deposition with HFA-BDP versus CFC-BDP (Fig 4). In normal subjects, the HFA-BDP MDI delivered 55 to 60% of the drug to the airways, with 29 to 30% deposited in the oropharynx, versus 4 to 7% pulmonary delivery and 90 to 94% oropharyngeal deposition with the CFC-MDI. Similar pulmonary delivery was noted with HFA-BDP in asthmatic subjects. Further, the distribution pattern seen with HFA-BDP was far more diffuse than that seen with CFC-BDP, where the drug was delivered primarily to the central airways. This authors concluded that this pattern reflected improved delivery of drug to the peripheral airways.18 The improved drug delivery seen as a result of these changes in MDI technology should allow improved targeting of the distal lung with inhaled corticosteroids and other pharmacologic agents and will provide new insights

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Figure 4. Differential pulmonary deposition of radiolabeled BDP with HFA-134a-BDP MDI (panel A) vs CFC-BDP MDI (panel B). Panels A and B are gamma scintigraphic images. Reprinted from Reference 18.

into the relationship between distal lung inflammation and the clinical manifestations of asthma. Cysteinyl Leukotriene Receptor Antagonists and Distal Lung Inflammation The cysteinyl leukotrienes (leukotrienes C4 [LTC4], LTD4, and LTE4) are thought to be important mediators of airway responsiveness, bronchoconstriction, mucus hypersecretion, and airway blood vessel permeability in asthma.19 –21 Cysteinyl leukotrienes appear to bind at least two receptors, al-

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though most of the biologic effects of cysteinyl leukotrienes are a result of binding of cysteinyl leukotriene-1 (CysLT1) receptor subtype.22 In 1999, Lynch et al23 reported that the CysLT1 receptor was a 337-amino acid G protein-coupled receptor which was expressed in the lung, spleen, and peripheral blood leukocytes and could be mapped to the long arm of the X chromosome.23 In 2001, Figueroa et al24 further characterized the CysLT1 receptor in the lung using immunohistochemical methods. They were able to show that the CysLT1 receptor mRNA was distributed throughout normal human lung tissue in airway smooth muscle fibers and that expression of the CysLT1 receptor mRNA coincided with the presence of the receptor protein. The CysLT1 mRNA and receptor protein were also identified in human lung interstitial macrophages, which were closely associated with the smooth muscle cells.24 Heise et al25 demonstrated a somewhat different expression of the CysLT2 receptor mRNA in normal human lung, with the strongest expression of the CysLT2 receptor seen in interstitial macrophages, with much weaker expression in airway smooth muscle cells. Drugs that specifically antagonize the binding of cysteinyl leukotrienes to these receptors (montelukast, pranlukast, zafirlukast) have become available in recent years and have been shown to have multiple anti-inflammatory effects.26 They are currently recommended as second-line or alternative controller medications (after inhaled corticosteroids) in the treatment of persistent asthma.13 Given the distribution of CysLT1 and CysLT2 receptors in the airway smooth muscle and interstitium as described above, there is significant potential for these agents to modify distal lung inflammation. Clinical trials that are designed to reveal the impact of these agents on small airway inflammation will be an important addition to our understanding of the evolving role of these drugs in asthma therapy. CONCLUSION There is mounting evidence that small airway inflammation plays an important role in the asthma clinical phenotype. However, much work remains to be done. Ongoing advances in research techniques such as transbronchial biopsy and morphometry will permit detailed analysis of the inflammatory changes in the small airways in asthma. The relationship between pathology and findings on HRCT will ultimately improve our ability to evaluate these changes noninvasively, and new drugs and methods of drug delivery will improve our targeting of this important lung compartment in asthma. REFERENCES 1. Wagner EM, Liu MC, Weinmann GG, et al. Peripheral lung resistance in normal and asthmatic subjects. Am Rev Respir Dis 1990;141:584 –588. 2. Irvin CG, Pak J, Martin RJ. Airway-parenchyma uncoupling in nocturnal asthma. Am J Respir Crit Care Med 2000;161:50 –56. 3. Carroll N, Elliot J, Morton A, et al. The structure of large and small airways in nonfatal and fatal asthma. Am Rev Respir Dis 1993;147:405– 410.

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4. Haley KJ, Sunday ME, Wiggs BR, et al. Inflammatory cell distribution within and along asthmatic airways. Am J Respir Crit Care Med 1998;158:565–572. 5. Kraft M, Djukanovic R, Wilson S, et al. Alveolar tissue inflammation in asthma. Am J Respir Crit Care Med 1996;154: 1505–1510. 6. Djukanovic R, Roche WR, Wilson JW, et al. Mucosal inflammation in asthma. Am Rev Respir Dis 1990;142:434 – 457. 7. Kraft M, Martin RJ, Wilson S, et al. Lymphocyte and eosinophil influx into alveolar tissue in nocturnal asthma. Am J Respir Crit Care Med 1999;159:228 –234. 8. King GG, Muller NL, Pare PD. Evaluation of airways in obstructive pulmonary disease using high-resolution computed tomography. Am J Respir Crit Care Med 1999;159:992–1004. 9. Okazawa M, Muller N, McNamara AE, et al. Human airway narrowing measured using high resolution computed tomography. Am J Respir Crit Care Med 1996;154:1557–1562. 10. Goldin JG, McNitt-Gray MF, Sorenson SM, et al. Airway hyperreactivity: assessment with helical thin-section CT. Radiology 1998;208:321–329. 11. Montreal Protocol. The Montreal Protocol on substances that deplete the ozone layer. In: Final Act (Nairobi: UNEP, 1987). Federal Register, Washington, DC: 1994; 56276 –56298. 12. Anderson PJ. Delivery options and devices for aerosolized therapeutics. Chest 2001;120(Suppl):89S–93S. 13. Guidelines for the diagnosis and management of asthma. Bethesda, MD: National Heart, Lung and Blood Institute, 1997. Report no 97– 4051. 14. Laube BL. In vivo measurements of aerosol dose and distribution: clinical relevance. J Aerosol Med 1996;9(Suppl):S77–S91. 15. Leach CL. Enhanced drug delivery through reformulating MDIs with HFA propellants-drug deposition and its effect on preclinical and clinical programs. In: Dalby RN, Byron PR, Farr SJ, editors. Respiratory Drug Delivery V Proceedings. Buffalo Grove: Interpharm Press, 1996:133–144. 16. Vanden Burgt JA, Busse WW, Martin RJ, et al. Efficacy and safety overview of a new inhaled corticosteroid, QVAR (hydrofluoroalkane-beclomethasone extrafine inhalation aerosol), in asthma. J Allergy Clin Immunol 2000;106:1209 –1226. 17. Gross G, Thompson PJ, Chervinsky P, et al. Hydrofluoroalkane134a beclomethasone dipropionate, 400 ␮g, is as effective as chlorofluorocarbon beclomethasone dipropionate, 800 ␮g, for the treatment of moderate asthma. Chest 1999;115:343–351. 18. Leach CL, Davidson PJ, Boudreau RJ. Improved airway targeting with the CFC-free HFA-beclomethasone metered-dose inhaler compared with CFC-beclomethasone. Eur Respir J 1998; 12:1346 –1353. 19. Adelroth E, Morris MM, Hargreave FE, et al. Airway responsiveness to leukotrienes C4 and D4 and to methacholine in patients with asthma and normal controls. N Engl J Med 1986; 315:480 – 484. 20. Dahlen SE, Hedqvist P, Hammarstrom S, et al. Leukotrienes are potent constrictors of human bronchi. Nature 1980;288: 484 – 486. 21. Lewis RA, Austen KF, Soberman RJ. Leukotrienes and other products of the 5-lipoxygenase pathway. Biochemistry and relation to pathobiology in human diseases. N Engl J Med 1990; 323:645– 655. 22. Metters KM. Leukotriene receptors. J Lipid Mediat Cell Signal 1995;12:413– 427. 23. Lynch KR, O’Neill GP, Liu Q, et al. Characterization of the

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human cysteinyl leukotriene CysLT1 receptor. Nature 1999;399: 789 –793. 24. Figueroa DJ, Breyer RM, Defoe SK, et al. Expression of the cysteinyl leukotriene 1 receptor in normal human lung and peripheral blood leukocytes. Am J Respir Crit Care Med 2001; 163:226 –233. 25. Heise CE, O’Dowd BF, Figueroa DJ, et al. Characterization of the human cysteinyl leukotriene 2 receptor. J Biol Chem 2000; 275:30531–30536.

26. Diamant Z, Sampson AP. Anti-inflammatory mechanisms of leukotriene modulators. Clin Exp Allergy 1999;29:1449 –1453. Requests for reprints should be addressed to: Richard J. Martin, MD 1400 Jackson Street Denver, CO 80206 E-mail: [email protected]

CME Examination 1–5, Sutherland ER and Richard J. Martin RJ. 2002;89:119-124. CME Test Questions 1. The distal lung is defined as: a. All airways distal to the mainstem bronchi. b. Airways with a diameter of ⬎2mm. c. Airways with a diameter of ⬍2 mm. d. Alveolar tissue. e. c and d. 2. Distal lung inflammation: a. Does not alter airways resistance. b. Has no effect on airway-parenchymal coupling. c. Results in nocturnal uncoupling of airway and parenchyma. d. Has no effect on lung volumes. e. Has no known relationship to respiratory system compliance. 3. Radiographic techniques for evaluating the distal lung include: a. Postero-anterior and lateral chest radiograph. b. HRCT. c. Barium swallow.

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d. Positron emission tomography scanning. e. Ventilation-perfusion scan. 4. Alveolar tissue inflammation: a. Is not an important issue in asthma. b. May be evaluated with transbronchial biopsy. c. Does not change more than 24 hours in subjects with nocturnal asthma. d. Is associated with an increase in alveolar tissue eosinophils. e. b and d. 5. Drugs conclusively shown to reduce small airway inflammation include: a. Inhaled corticosteroids. b. Cysteinyl leukotriene receptor antagonists. c. Theophylline. d. Cromolyn. e. None of the above. Answers found on page 211.

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