Bronchial hyperresponsiveness is generally assessed by

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1 Provocation With Adenosine 5 -Monophosphate Increases Sputum Eosinophils* Maarten van den Berge, MD; H.A.M. Kerstjens, PhD; D.M. de Reus; H.F. Kauffman, PhD; G.H. Koëter, PhD; and D.S. Postma, PhD (CHEST 2003; 123:417S) Abbreviations: AMP adenosine 5 -monophosphate; MCh methacholine; PC 20 provocative concentration of a substance causing a 20% fall in FEV 1 Bronchial hyperresponsiveness is generally assessed by testing with methacholine (MCh) or histamine. Another possible stimulus with which to measure bronchial hyperresponsiveness is adenosine 5 -monophosphate (AMP). We have demonstrated previously 1 that the provocative concentration of a substance (ie, AMP) causing a 20% fall in FEV 1 (PC 20 ) better reflects airway inflammation in asthma patients than does the MCh PC 20. AMP, and by implication adenosine, acts by the release of a variety of inflammatory mediators from mast cells. We intended to assess with the study whether this AMPinduced mediator release initiates an inflammatory process resulting in the recruitment of eosinophils to the airways. Materials and Methods We included 21 nonsmoking atopic patients with asthma (mean FEV 1, 103% of predicted; mean age, 33 years). Eleven patients received therapy with inhaled corticosteroids. Each subject performed three sputum inductions on different days at least 7 days apart, one without previous provocation (ie, baseline), 1 h after the MCh PC 20 and 1 h after the AMP PC 20. Results The percentage of sputum eosinophils significantly increased after the AMP PC 20, but not after the MCh PC 20 (p 0.01 and 0.50, respectively) [Table 1]. This increase was independent of the use of inhaled corticosteroids (p 0.73). Discussion AMP provocation leads to increased sputum eosinophilia compared to MCh provocation. Interestingly, adenosine levels are elevated in the BAL fluid of asthmatic *From the Departments of Pulmonology (Drs. van den Berge, Kerstjens, Koëter, and Postma) and Allergology (Drs. de Reus and Kauffman), University Hospital Groningen, Groningen, the Netherlands. This research was supported by the University of Groningen and GlaxoSmithKline. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians ( permissions@chestnet.org). Correspondence to: Maarten van den Berge, MD, Department of Pulmonology, University Hospital Groningen, Groningen, the Netherlands; m.van.den.berge@int.azg.nl Table 1 Sputum Inflammatory Marker Levels After Control Visit and After AMP and MCh Challenges Sputum Cell Counts, % Without Previous Provocation patients, and they increase further after allergen challenge. Together with our findings, this indicates a role for adenosine in the development and maintenance of airway inflammation. Thus, the role of adenosine in asthma deserves further study as it may have clinical consequences for the treatment of asthma. Reference 1 h After PC 20 AMP 1 van den Berge M, Meijer RJ, Kerstjens HA, et al. PC 20 adenosine 5 -monophosphate is more closely associated with airway inflammation in asthma than PC 20 methacholine. Am J Respir Crit Care Med 2001; 163: Airway Remodeling in Asthma* Antonio M. Vignola, MD, PhD; Franco Mirabella, MD; Giorgio Costanzo, PhD; Rossana Di Giorgi, PhD; Mark Gjomarkaj, MD; Vincenzo Bellia, MD, FCCP; and Giovanni Bonsignore, MD, FCCP 1 h After PC 20 MCh Total Eosinophils Neutrophils Macrophages Lymphocytes Epithelial cells *Values given as mean SEM. p 0.01 (compared to baseline). Chronic inflammation and remodeling may follow acute inflammation or may begin insidiously as a low-grade smoldering response, especially in the case of immune reactions. The histologic hallmarks of chronic inflammation and remodeling are as follows: (1) infiltration by macrophages and lymphocytes; (2) proliferation of fibroblasts that may take the form of myofibroblasts; (3) angiogenesis; (4) increased connective tissue (fibrosis); and (5) tissue destruction. It is clear that changes in the extracellular matrix, smooth muscle, and mucous glands have the capacity to influence airway function and reactivity in asthma patients. However, it is not known how each of the many structural changes that occur in the airway wall contributes to altered airway function in asthma. In asthma, remodeling is almost always present in biopsy specimens (eg, collagen deposition on basement membrane) but is not always clinically demonstrated. Destruction and subsequent remodeling of the normal bronchial architecture are manifested by an accelerated decline in FEV 1 and bronchial hyperresponsiveness. This irreversible CHEST / 123 / 3/ MARCH, 2003 SUPPLEMENT 417S

2 component of airway obstruction is more prominent in patients with severe disease and even persists after aggressive anti-inflammatory treatment. Airway remodeling appears to be of great importance for understanding the long-term follow-up of asthmatic patients, but there are major gaps in our knowledge. Physiologic correlations with pathology represent a major missing link that should be filled. More long-term studies are needed to appreciate the prevention and treatment of remodeling. Future research therefore should provide better methods for limiting airway remodeling in asthma patients. (CHEST 2003; 123:417S 422S) Abbreviations: ECM extracellular matrix; EGF epidermal growth factor; IL interleukin; MMP matrix metalloproteinase; TGF transforming growth factor; TIMP tissue inhibitor of matrix metalloproteinase Although asthma has been considered as a condition of reversible airflow obstruction, many asthmatic patients, both children and adults, have evidence of residual airway obstruction that may even be detected in asymptomatic patients. This clinically demonstrable irreversible component of airways obstruction also is reflected by the presence of structural changes of the bronchi that contribute to geometric changes of the airways and to a various degree of functional impairment. 1 Remodeling is a critical aspect of wound repair in all organs representing a dynamic process that associates matrix production and degradation in reaction to an inflammatory insult, 2 leading to a normal reconstruction process or a pathologic process. Structural remodeling in airway diseases was proposed initially to describe changes induced in endothelial cells and extracellular matrix (ECM) due to an injury of the pulmonary circulation. It was then extended to many other pathologic situations including asthma. 3 Demonstration of Airway Remodeling in Asthma Various structural alterations have been described in the airways of asthmatic patients, 1 all of which can contribute to an overall increase in airway wall thickness. 4 Thickening of the Reticular Basement Membrane Thickening of the reticular basement membrane (ie, the lamina reticularis) [Figs 1 and 2] is a characteristic feature of the asthmatic bronchus, occurring early on in the disease process. It appears to consist of a plexiform *From the Istituto di Medicina Generale e Pneumologia (Drs. Vignola and Bellia) Cattedra di Malattie Respiratorie, Università di Palermo, Palermo, Italy; and Istituto di Biomedicina e Immunologia Molecolare (Drs. Vignola, Mirabella, Costanzo, Di Giorgi, Gjomarkaj, and Bonsignore), CNR, Palermo, Italy. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians ( permissions@chestnet.org). Correspondence to: A.M. Vignola, MD, PhD, Istituto di Medicina Generale e Pneumologia, Cattedra di Malattie Respiratorie, Università di Palermo, Via Trabucco 180, 90146, Palermo, Italy; vignola.am@iol.it deposition of Ig, collagen I and III, tenascin, and fibronectin, 5 but not of laminin. These proteins are likely produced by activated myofibroblasts, 6 leading to a so-called subepithelial fibrosis. In some studies, the thickening could not be related to the severity, duration, or etiology of asthma, 7 whereas in others a correlation with this and severity has been observed. 8 Thickening of the subepithelial layer also has been found in patients with cough-variant asthma, 9 in which, similar to the case with classic asthma, the patient presents with bronchoalveolar and bronchial tissue eosinophilia. Interstitial Matrix The remodeling processes of the interstitial matrix are less well-documented than the thickening of the lamina reticularis. However, many features can be observed. Elastic Fibers: The presence of an abnormal superficial elastic fiber network has been described in asthmatic subjects. Indeed, in bronchial biopsy specimens fibers appear to be fragmented or even absent, suggesting that an abnormal elastolytic process may occur in asthma subjects. 10 It is of note that even the deeper layer of elastic fibers is altered in most asthmatic patients, with the fibers often being patchy, tangled, and thickened, similar in appearance to solar elastolysis in the skin. Transmission electron microscopy studies have shown that an elastolytic process occurs in asthmatic patients. 10 In fatal cases of asthma, elastic fiber fragmentation also has been found in central airways and was associated with marked elastolysis. 11 It seems, however, that changes in elastic fibers are not due solely to increased degradation but may also be influenced by the deposition of other matrix proteins, such as collagen. Indeed, in the bronchial tree elastic fibers, collagen and myofibroblast matrix structures form submucosal longitudinal bundles, which appear to be hypertrophied as a result of an increased amount of collagen and myofibroblast matrix deposition. 12 It appears, therefore, that the increased level of elastolysis in asthma patients is part of a more complex process that, regulating in a dynamic fashion the size of such a submucosal network, may have not only a great impact on the geometry and distensibility of the airways, but also may affect airway function. 12 Collagen and Proteins: In the submucosa of asthmatic patients, electron microscopy studies show that collagen is not completely normal. In some patients hyperplasia of collagen fibers can be observed. 12 Fibronectin, laminin and tenascin deposition in the submucosa was found in the biopsies of asthmatics but not in those of healthy subjects (Fig 1). 1 Fibronectin levels are increased in the BAL fluid of asthmatic patients and correlate with the levels of immunoreactive transforming growth factor (TGF)-. 13 Blood Vessels Airway wall remodeling in asthma patients involves a number of changes, including increased vascularity, vasodilatation, and microvascular leakage. Evidence suggests 418S Thomas L. Petty 45th Annual Aspen Lung Conference: Asthma in the New Millennium

3 Figure 1. Cascade of events following the injury of the bronchial epithelium and their potential impact on tissue remodeling. 1: As a consequence of the loss of epithelium integrity due, at least partly, to apoptosis, most of the ciliated bronchial epithelial cells undergo desquamation. 2: On the other hand, basal epithelial cells remaining attached to the basement membrane express high levels of Bcl-2, an antiapoptotic molecule, in the attempt to initiate a repair cycle. 3: This process, however, may not be accomplished due to a reduced ability of basal cells to undergo proliferation (proliferating cell nuclear antigen). 4: In addition, basal epithelial cells appear to be activated (increased nuclear factor-kb expression) and release several mediators involved in inflammation and repair, including TGF-. Asa result of this vicious circle, the epithelium becomes a source of mediators (ie, cytokines and growth factors) that perpetuates the damage of the epithelium and promotes the exaggerated deposition of matrix proteins and the development of airway remodeling. that the number and size of bronchial vessels is moderately increased in patients with asthma compared with normal controls (for a review, see Vignola et al 1 and Bousquet et al 3 ). In particular, there may be increased numbers of vessels in patients with fatal asthma, 14 but the extent of neovascularization or angiogenesis is still unclear. Smooth Muscle Hypertrophy and hyperplasia of airway smooth muscle 15 have been reported in postmortem specimens of asthmatic bronchi. In patients who died from an asthma exacerbation, the increase in smooth muscle was far greater than in those who died from another cause. 15 In vivo animal studies have confirmed that prolonged allergen exposure can increase smooth muscle. 16 Mucous Glands Mucous glands are distributed throughout the airways of asthma patients and, in the form of goblet cells, are even present in peripheral bronchioles where normally they are absent. The mucous glands in the segmental bronchi of asthmatic patients are considerably enlarged, and Dunnill et al 17 showed that the volume of mucous glands was twice as great in asthmatic patients compared to healthy subjects. Animal models 16 have indicated that goblet cell metaplasia and increased mucus production can occur in the large airways following prolonged allergen exposure, suggesting that a persistent inhalation of allergens may be an important causative factor for the development of mucus gland remodeling. Biology of Airway Remodeling The Epithelium-Mesenchymal Interactions: In asthma patients, the bronchial epithelium is highly abnormal, with structural changes involving the separation of columnar cells from their basal attachments, and functional changes including increased expression and release of proinflammatory cytokines, growth factors, and mediatorgenerating enzymes. Beneath this damaged structure, CHEST / 123 / 3/ MARCH, 2003 SUPPLEMENT 419S

4 Figure 2. Increased thickness of the basement membrane (arrow), and enhanced and abnormal deposition of fibronectin, which is identified by immunohistochemistry using a specific monoclonal antibody (dashed arrow), in the bronchial submucosa. there is an increased number of subepithelial myofibroblasts that deposit interstitial collagens, causing thickening and increased density of the subepithelial basement membrane. A failure in the injury-repair cycle of damaged epithelial cells seems to play an important role in the development of abnormal epithelium-mesenchymal interactions. Some data 18 show that in asthma patients residual epithelial cells that have not undergone desquamation do not show a greater propensity to undergo apoptosis than epithelial cells undergoing apoptosis, a feature that may ensure the development of the repair-injury cycle. Interestingly, the epithelial cells of asthmatic patients also are characterized by a high immunoreactivity for Bcl-2 and Hsp27, two molecules that are characterized by both protective and antiapoptotic properties, suggesting that bronchial epithelial cells of asthmatic subjects have the ability to protect themselves against inflammatory stimuli (Fig 1). 18 In addition, it appears that the bronchial epithelium of untreated asthmatic subjects expresses low levels of proliferating markers, such as proliferating cell nuclear antigen, 18 despite extensive damage, 3,19 revealing a potential failure in the epithelial injury-repair cycle in response to local inflammatory and inhaled agents. Moreover, injury of the epithelium in asthma patients results in a localized and persistent increase in epidermal growth factor (EGF) receptor immunostaining at the wound edge, a mechanism that may cause the epithelium to be locked in a repair phenotype. 20 It is known that during repair, cells produce a wide range of substances, such as growth factors, that are capable of modulating the wound repair process. Thus, the blockage of the epithelium in a repair phenotype with a low proliferative rate establishes a vicious circle that is sustained by the continuous release of growth factors and other profibrotic mediators (such as TGF-, fibroblast growth factor, endothelin, and EGF) [Fig 2] that directly regulate the phenotypic and functional features of mesenchymal cells that are located underneath the basement membrane, such as fibroblasts and myofibroblasts. 19 The increased expression of insulin-like growth factor-1, EGF, and TGF- in biopsy specimens or BAL fluid samples has been reported. 1 TGF- expression was found to correlate with the degree of subepithelial fibrosis 21 and to be significantly increased in patients with severe asthma who had a rich eosinophilic infiltration of the airways. 22 Under the effects of epithelial-derived growth factors, mesenchymal cells produce collagen, reticular and elastic fibers, as well as proteoglycans and glycoproteins of the amorphous ECM, all of which likely contribute to the thickened airway wall of asthmatic subjects. These consequences also are demonstrated by the enhanced proteoglycan deposition in the subepithelial layer of the airway wall of asthmatic subjects. 23 It is therefore likely that ECM production and deposition is under the control of the epithelial-mesenchymal unit, leading to the development of structural alterations that are topographically distributed in the inner layer (ie, the tissue between the luminal surface and the smooth muscle layer). This process may have dramatic functional consequences as the degree of luminal effacement in response to a given stimulus for smooth muscle contraction is augmented by an increase in the volume of the inner airway wall. In addition to growth factors, remodeling processes occurring at the level of the inner airway wall are regulated by some cytokines such as interleukin (IL)-11. IL-11 is a 420S Thomas L. Petty 45th Annual Aspen Lung Conference: Asthma in the New Millennium

5 pleiotropic cytokine, the expression of which has been found 24 to be increased in the bronchial epithelial cells of asthmatic subjects. Targeted overexpression of this cytokine in mice has resulted in the remodeling of the airways and in the development of airway hyperresponsiveness and airway obstruction. In addition, when compared with control animals, IL-11 transgenic mice develop an enhanced inner wall thickness that is associated with airways obstruction and increased airway responses to methacholine. 25 Mast Cells and Fibroproliferative Response In addition to epithelial cells, the activation of mesenchymal cells can be modulated by other cell types, such as mast cells. 19 Recently, an increased number of tryptasepositive mast cells was found in the bundles of airway smooth muscle from subjects with asthma, suggesting that the infiltration of airway smooth muscle by mast cells is associated with the disordered airway function in asthma patients. 26 One mediator that is released in high concentrations from degranulating mast cells is tryptase. This serine protease is a potent stimulant of fibroblast and smooth muscle cell proliferation, and it is capable of stimulating synthesis of type I collagen by human fibroblasts. 27 A major mechanism involved in the regulation of fibroblast proliferation appears to be the cleavage and activation of protease-activated receptor-2, which is expressed on the cell surface of lung fibroblasts. 27 Evidence has suggested 28 that mast cells also may influence the development of airway remodeling in asthma patients by releasing high amounts of plasminogen activator inhibitor type 1. Fibroblasts and Myofibroblasts Fibroblasts and myofibroblasts can contribute to tissue remodeling by releasing ECM components such as elastin, fibronectin, and laminin. 1 Increased numbers of myofibroblasts are found in the airways of asthmatic patients, and their number appears to correlate with the size of the basement reticular membrane. 19 Following bronchial allergen challenge, myofibroblast numbers are increased, the cells undergo a differentiation process, and present with structural and ultrastructural features that are similar to those of smooth muscle cells. 29 These latter cells, in addition to contractile responses and mitogenesis, have synthetic and secretory potentials, as they are regulated by activation, normal T-cell expressed and secreted (or RANTES). They may participate in chronic airway inflammation by interacting with cytokines derived from both T helper type 1 and T helper type 2 cells to modulate chemoattractant activity for eosinophils, activated T lymphocytes, and monocytes/macrophages. 30 Smooth Muscle Cells In patients who died from an asthma exacerbation, the increase in smooth muscle was far greater than in those who died from another cause, and in vivo animal studies have confirmed that prolonged allergen exposure can increase smooth muscle thickness. 16 Cell culture studies have disclosed a wide range of soluble factors that can promote the proliferation of human airway smooth muscle cells, suggesting that, through autocrine loops, these cells can regulate their own proliferative rate. Evidence 31 has shown the presence of smooth muscle mitogens in BAL fluid samples from asthmatic subjects who have undergone allergen challenge. Holgate et al 19 and Naureckas et al 31 have shown that EGF activates ErbB-2 and stimulates phosphatidylinositol 3-kinase in human airway cells, which is a mechanism that induces transcription from the cyclin D1 promoter, thereby influencing an important checkpoint of cell proliferation. An additional mechanism regulating smooth muscle proliferation is represented by the production of matrix metalloproteinase (MMP)-2, which has been demonstrated 32 to be an important autocrine factor that is required for airway smooth muscle proliferation. Proteases and Protease Inhibitors MMPs selectively degrade ECM components. MMPs also play a crucial role in the trafficking of inflammatory and structural cells. Imbalances between MMPs and their inhibitors may contribute to tissue damage and some of the remodeling features seen in asthma. MMP-9 can degrade native type IV and type V collagens, denatured collagens, entactin, proteoglycans, and elastin. An excess of tissue inhibitor of matrix metalloproteinase (TIMP)-1 over MMP-9 in stable asthma patients may interfere with cell trafficking and tissue repair, and also may contribute to increased ECM deposition and fibrosis by the inhibition of MMP-9 or other MMPs in vivo by ECM. By contrast, an increase in MMP-9 resulting in a high MMP-9/TIMP-1 ratio has been demonstrated in patients with acute asthma 33 or following allergen challenge, 34 suggesting that ECM and basement membrane degeneration by MMPs and other proteinases may occur in the early phase of asthma exacerbation, and that TIMP-1 production may accompany or follow the MMP-9 increase to inhibit these processes. Conclusion The presence of structural changes in the airways of asthmatic patients has clearly been established. It is equally clear that this remodeling process could have a profound influence on airway behavior. It also appears that airway inflammation and remodeling are interdependent processes that clearly influence the clinical long-term evolution of asthma. Thus, understanding the cellular and molecular mechanisms underlying airway remodeling is of great importance for the development of new and effective therapeutic strategies for asthma patients. References 1 Vignola AM, Kips J, Bousquet J. Tissue remodeling as a feature of persistent asthma. J Allergy Clin Immunol 2000; 105: Cotran R, Kumar V, Robin S. Inflammation and repair. Robbins Pathologic Basis of Disease 1989; Bousquet J, Jeffery PK, Busse WW, et al. Asthma: from CHEST / 123 / 3/ MARCH, 2003 SUPPLEMENT 421S

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Sputum matrix metalloproteinase-9: tissue inhibitor of metalloproteinase-1 ratio in acute asthma. J Allergy Clin Immunol 2000; 105: Kelly EA, Busse WW, Jarjour NN. Increased matrix metalloproteinase-9 in the airway after allergen challenge. Am J Respir Crit Care Med 2000; 162: Interleukin-13 Stimulates the Proliferation of Lung Myofibroblasts via a Signal Transducer and Activator of Transcription-6-Dependent Mechanism* A Possible Mechanism for the Development of Airway Fibrosis in Asthma Jennifer L. Ingram, PhD; Annette Rice, BS; Kristen Geisenhoffer, BS; David K. Madtes, MD; and James C. Bonner, PhD (CHEST 2003; 123:422S 424S) Abbreviations: IL interleukin; PDGF platelet-derived growth factor; STAT signal transducer and activator of transcription; TGF transforming growth factor 422S Thomas L. Petty 45th Annual Aspen Lung Conference: Asthma in the New Millennium