Pathogenic Mechanisms in Asthma and COPD

5
PART

Pathophysiology of Asthma
INTRODUCTION
This chapter aims to provide a brief overview of disease mechanisms to integrate some of the detailed information provided in earlier chapters into a framework with clinical relevance. Asthma and chronic obstructive pulmonary disease (COPD) are both highly complex conditions whose pathobiology remains inadequately dened. In both diseases there are many dierent and distinct inammatory cells and multiple mediators with complex acute and chronic eects on the airways and airspaces. We now appreciate that these changes may vary among patients because of genetic variance in susceptibility [1]. Although our understanding of asthma pathogenesis has been enhanced by the application of new molecular, cell biological, and genetic techniques to the condition, and the key test of the acquired knowledge is the success of new specically targeted therapies. In this area the successes have been few in asthma and are virtually absent in COPD. Thus, even though we have made considerable advances in understanding asthma, there remain many fundamental questions that need to be answered. Our views on asthma and COPD have continued to evolve. Although it is recognized that chronic inammation underlies the clinical syndromes, it has been hard to dene the precise nature of this inammation, much less its primary etiology. Nevertheless it is appreciated that the nal consequence of this chronic inammatory response is an abnormal control of airway caliber in vivo in asthma. In contrast, the evolution of understanding in COPD has been much slower; key insights are sought which will open up the understanding of this complex disorder (see Chapter 34).

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CHAPTER
Peter J. Barnes1 and Jeffrey M. Drazen2
National Heart and Lung Institute (NHLI), Clinical Studies Unit, Imperial College, London, UK 2 Department of Medicine, Pulmonary Division, Brigham and Womens Hospital, Boston, MA, USA
1

ASTHMA AS AN INFLAMMATORY DISEASE

It had been recognized for many years that patients who die of asthma attacks have grossly inamed airways. The airway lumen is occluded by tenacious mucus plugs composed of plasma proteins exuded from airway vessels and mucus glycoproteins secreted from surface epithelial cells. The airway wall is edematous and inltrated with inammatory cells, which are predominantly eosinophils and T-lymphocytes [2]. The airway epithelium is invariably shed in a patchy manner and clumps of epithelial cells are found in the airway lumen. Occasionally there have been opportunities to examine the airways of asthmatic patients who die in accidents thought to be unrelated to their asthma. In this setting inammatory changes have been observed but they are less marked than those observed in patients with active asthma [3]. These studies also reveal that the inammation in asthmatic airways involves not only the trachea and bronchi, but also extends to the terminal bronchioles [4]; some investigators using transbronchial biopsies have shown inammatory cells in the parenchyma [5]. It has also been possible to examine the airways of asthmatic patients by beroptic and rigid bronchoscopy, bronchial biopsy, and bronchoalveolar lavage (BAL). Direct bronchoscopic examination reveals that the airways of asthmatic patients are often erythematous and swollen, indicating acute inammation. Lavage of the airways has revealed an increase in the numbers of lymphocytes, mast cells, and eosinophils and evidence for activation of macrophages in comparison with nonasthmatic controls. Biopsies have provided evidence for increased numbers and activation of mast cells, macrophages, eosinophils,

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Asthma and COPD: Basic Mechanisms and Clinical Management T-lymphocytes including regulatory T-cells, and mucin secreting cells [69]. These changes are found even in patients with mild asthma who have few symptoms [10], suggesting that inammation may be found in all asthmatic patients who are symptomatic. Similar inammatory changes have been found in bronchial biopsies of children with asthma, even at the onset of symptoms [11, 12]. airway cells or that modify the mechanical characteristics of the airway wall. AHR may be due to increased release of mediators (such as histamine and leukotrienes from mast cells), abnormal behavior of airway smooth muscle, thickening of the airway wall by reversible (edema), and irreversible (airway smooth muscle thickening, brosis) elements. Airway sensory nerves may also contribute importantly to symptoms, such as cough and chest tightness, as the nerves become sensitized by the chronic inammation in the airways. Although most attention has been focused on the acute inammatory changes seen in asthmatic airways (bronchoconstriction, plasma exudation, mucus secretion), asthma is a chronic disease, persisting over many years in most patients. Superimposed on this chronic inammatory state are acute inammatory episodes, which correspond to exacerbations of asthma. It is clearly important to understand the mechanisms of acute and chronic inammation in asthmatic airways and to investigate the long-term consequences of this chronic inammation on airway function.

AIRWAY HYPERRESPONSIVENESS
The relationship between inammation and clinical symptoms of asthma is not clear. There is evidence that the degree of inammation is loosely related to airway hyperresponsiveness (AHR), as measured by histamine or methacholine challenge. However, the degree of inammation, as measured by the number of various types of inammatory cells in the lesion, is not closely linked to the clinical severity of asthma or AHR [13]. This suggests that other factors, such as structural changes in the airway wall, are important in mediating the clinical symptoms of asthma. The increased airway responsiveness in asthma is a striking physiological abnormality that is present even when airway function is otherwise normal. It is likely that there are several factors that underlie this increased responsiveness to constrictor agents, particularly those that act indirectly by releasing bronchoconstrictor mediators from

INFLAMMATORY CELLS
Many dierent inammatory cells are involved in asthma, although the precise role of each cell type is not yet certain (Fig. 33.1). It is evident that no single inammatory cell is
Inhaled allergens

Epithelial cells CCL11 Mast cell Histamine Cys-LTs PGD2 IL-9 CCL17 CCL22 Bronchoconstriction IgE IL-4, IL-13 CCR4 Th2 cell IL-5 CCR3 T-reg SCF TSLP Dendritic cell

B-Iymphocyte Eosinophils
FIG. 33.1 A potential schema of Inflammation in asthma. Inhaled allergens activate sensitized mast cells by crosslinking surface-bound IgE molecules to release several bronchoconstrictor mediators, including cysteinyl-leukotrienes (cys-LT) and prostaglandin D2 (PGD2). Epithelial cells release stem-cell factor (SCF), which is important for maintaining mucosal mast cells at the airway surface. Allergens are processed by myeloid dendritic cells, which are conditioned by thymic stromal lymphopoietin (TSLP) secreted by epithelial cells and mast cells to release the chemokines CC-chemokine ligand 17 (CCL17) and CCL22, which act on CC-chemokine receptor 4 (CCR4) to attract T helper 2 (Th2) cells. Th2 cells have a central role in orchestrating the inflammatory response in allergy through the release of interleukin-4 (IL-4) and IL-13 (which stimulate B-cells to synthesize IgE), IL-5 (which is necessary for eosinophilic inflammation) and IL-9 (which stimulates mast-cell proliferation). Epithelial cells release CCL11, which recruits eosinophils via CCR3. Patients with asthma may have a defect in regulatory T-cells (T-reg), which may favor further Th2-cell proliferation.

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Pathophysiology of Asthma able to account for the complex pathophysiology of asthma but some cells predominate in asthmatic inammation. The inammation in asthmatic airways diers strikingly from that observed in COPD, where there is a predominance of macrophages, cytotoxic (CD8) T-lymphocytes, and neutrophils, although both of these common diseases may coexist in some patients. However, although the dierences in inammation between mild asthma and COPD are well described, it is not recognized that patients with severe asthma have inammatory changes that are similar to those of COPD, with increased numbers of neutrophils, CD4 Th1 and CD8 cells [14].

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Macrophages
Macrophages, which are derived from blood monocytes, may trac into the airways in asthma and may be activated by allergen via low-anity IgE receptors (FcRII) [23]. The enormous immunological repertoire of macrophages allows these cells to produce many dierent products, including a large variety of cytokines that may orchestrate the inammatory response. Macrophages have the capacity to initiate a particular type of inammatory response via the release of a certain pattern of cytokines. Macrophages may increase or decrease inammation depending on the stimulus. For example, alveolar macrophages from normal and patients with mild asthma phagocytose apoptic cells resulting from lipopolysaccharide (LPS) exposure in vitro and produce the anti-inammatory mediator PGE2. In contrast alveolar macrophages from patients with severe asthma are impaired with respect to apoptotic cell uptake and production of PGE2 [24]. These observations indicate that the severity of asthma and perhaps its chronicity can modulate the role of macrophages in asthma. It is also possible that the opposite is true, that is, patients with severe asthma are the individuals with defective macrophage function. It is established that repeated short term exposure to ozone recruits alveolar macrophages to the airways of patients with mild asthma [25] and that there is an association between the amount of carbon in alveolar macrophages, which had to be activated to ingest this material, and lung function [26]. Thus the role of alveolar macrophages appears to be to modulate the expression and severity of asthma.

Mast cells
Mast cells are clearly important in initiating the acute bronchoconstrictor responses to allergen and probably to other indirect stimuli, such as exercise and hyperventilation (via osmolality or thermal changes) and fog. Mast cell activation is likely to play a key role in the symptoms of asthma and during acute exacerbations. There is inltration of airway smooth muscle by mast cells in patients with asthma that is associated with the characteristic disordered airway function of this condition [15]. Treatment of asthmatic patients with prednisone results in a decrease in the number of tryptase-only positive mast cells [16]. Furthermore, the number of mast cells in induced sputum in patients with seasonal allergic rhinitis is related to the degree of AHR [17]. There appears to be an inltration of mast cells into airway smooth muscle of asthmatic patients and this is associated with AHR [15]. The key role of mast cells in asthma has been the subject of comprehensive reviews [7, 18]. Mast cells are maintained at the mucosal surface by the secretion of stem cell factor, which acts on c-kit receptors, by epithelial cells [19]. However, it is now clear that mast cells alone cannot account for all the pathobiological changes in asthma and that other cells, such as macrophages, eosinophils, and T-lymphocytes, play key roles in the chronic inammatory process including induction and maintenance of AHR. Classically mast cells are activated by allergens through an IgE-dependent mechanism. The importance of IgE in the pathophysiology of asthma has been highlighted by clinical studies with humanized anti-IgE antibodies, which inhibit IgE-mediated eects. Although anti-IgE antibody results in a reduction in circulating IgE to undetectable levels, this treatment results in minimal clinical improvement in patients with severe steroid-dependent asthma [20]. Examination of the patients receiving anti-IgE therapy with omalizumab reveals downregulated expression of IgE receptors (FcRI) on mast cells and basophils [21]. Biopsy studies show a profound reduction in tissue eosinophilia, together with reductions in T-cell and B-cell numbers in the airway submucosa [22]. These observations suggest that treatment with anti-IgE may be more eective in early stage rather than late stage asthma; however, no clinically directive trials with omalizumab have been conducted in this population. Furthermore, unless the cost of such therapy is dramatically reduced it is unlikely that such treatment would be used in patients with mild disease.

Dendritic cells
By contrast, dendritic cells (which are specialized macrophage-like cells in the airway epithelium) are very eective antigen-presenting cells and may therefore play a very important role in the initiation of allergen-induced responses in asthma [27, 28]. Dendritic cells take up allergens though their long nger-like projections, which extend into the airway lumen, process them to peptides, and migrate to local lymph nodes where they present the allergenic peptides to uncommitted T-lymphocytes, to program the production of allergen-specic T-cells. Although there is no established ways to manipulate dendritic cells, it has been shown that during experimental allergen challenge in humans that a signicantly greater proportion of CD33 cells expressed the cys-LT1-receptor compared with CD123 cells. Pranlukast, cys-LT1-receptor antagonist, decreased the allergen-induced decrease in CD33 dendritic cells at 3 h compared with placebo treatment. Based on this nding it is reasonable to speculate that anti-leukotriene treatment could modify the initial responses to allergen exposure [29].

Eosinophils
Eosinophil inltration is a characteristic feature of asthmatic airways and dierentiates asthma from other non-infectious inammatory conditions of the airway. Indeed, asthma was described as chronic eosinophilic bronchitis as early as 1916.
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Asthma and COPD: Basic Mechanisms and Clinical Management Allergen inhalation results in a marked increase in eosinophils in BAL uid at the time of the late reaction, and there is a correlation between eosinophil counts in peripheral blood or bronchial lavage and AHR. Eosinophils are linked to the development of AHR through the release of basic proteins and oxygen-derived free radicals [30, 31]. An important area of research is now concerned with the mechanisms involved in recruitment of eosinophils into asthmatic airways. Eosinophils are derived from bone marrow precursors; the Th2 cytokine IL-5 is a unique mediator of eosinophil dierentiation and survival in response to allergen provocation. After allergen challenge eosinophils appear in BAL uid during the late response, and this appearance is associated with a decrease in peripheral eosinophil counts and with the appearance of eosinophil progenitors in the circulation. The signal for increased eosinophil production is likely IL-5 derived from the inamed airway. Eosinophil recruitment initially involves adhesion of eosinophils to vascular endothelial cells in the airway circulation, their migration into the submucosa, and their subsequent activation. The role of individual adhesion molecules, cytokines, and mediators in orchestrating these responses has been extensively investigated but since there are no pathways that are unique to eosinophils, this aspect of eosinophil biology has not been extensively probed. Adhesion of eosinophils involves the expression of specic glycoprotein molecules on the surface of eosinophils (integrins) and their expression of such molecules as intercellular adhesion molecule-1 (ICAM-1) on vascular endothelial cells. An antibody directed at ICAM-1 markedly inhibits eosinophil accumulation in the airways after allergen exposure and also blocks the accompanying hyperresponsiveness [32]. However, ICAM-1 is not selective for eosinophils and cannot account for the selective recruitment of eosinophils in allergic inammation. Eosinophil migration may be due to the eects of lipid mediators, such as leukotrienes [33, 34] and possibly platelet-activating factor (PAF), to the eects of cytokines, such as GM-CSF and IL5 both of which are important for the survival of eosinophils in the airways and prime eosinophils to exhibit enhanced responsiveness. Eosinophils from asthmatic patients show exaggerated responses to PAF and phorbol esters, compared with eosinophils from atopic nonasthmatic individuals [35]. This is further increased by allergen challenge [36, 37] suggesting that they may have been primed by exposure to cytokines in the circulation. There are several mediators involved in the migration of eosinophils from the circulation to the surface of the airway. There are data showing that cysteinyl leukotrienes, through stimulation at the cys-LT1-receptor, are in part responsible for eosinophilopoiesis in human airways [38], but other mediators such as the eotaxins, a family of chemoattractant cytokines that promote eosinophil recruitment to tissues in response to allergic provocation via CCR3 receptors also play a role [3942]. Other potent and selective eosinophil chemoattractants include chemokines, such as CCL5 (RANTES) and CCL13 (MCP-4), that are expressed in epithelial cells [40, 43, 44] but data for their involvement in intact humans are minimal. There appears to be a co-operative interaction between IL-5, IL-13, and chemokines, so that more than one cytokine may be necessary for the eosinophilic response in airways [45, 46]. Once recruited to the airways, eosinophils
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require the presence of various growth factors, of which GM-CSF and IL-5 appear to be the most important [47]. In the absence of these growth factors eosinophils undergo programmed cell death (apoptosis) [48]. A humanized monoclonal antibody to IL-5 has been administered to asthmatic patients [49]; and, as in animal studies, there is a profound and prolonged reduction in circulating eosinophils. Although the inltration of eosinophils into the airway after inhaled allergen challenge is completely blocked, there is no eect on the response to inhaled allergen and no reduction in AHR. Anti-CCR3, anti-integrin, and antiselectin treatments are currently being considered as anti-eosinophil strategies for treatment of asthma; the results of these studies should better dene the role of eosinophils as mediators of asthmatic events. Eosinophils may play a role in airway remodeling rather than AHR in asthma as anti-IL-5 treatment is able to reduce extracellular matrix deposition in asthma [50].

Neutrophils
The eosinophil has been the recipient of current attention as an eector cell in asthma, but attention has been returning to the role of neutrophils [51, 52]. Although neutrophils are not a predominant cell type observed in the airways of most patients with mild-to-moderate chronic asthma, they appear to be a more prominent cell type in airways and induced sputum of patients with more severe asthma [5357]. The mechanism of neutrophilic inammation are not yet well understood, but several mediators, including CXCL8 (IL-8) are likely to be involved (Fig. 33.2). Sputum analysis shows that 20% of asthmatics, even with mild disease, have a predominantly neutrophilic pattern of inammation [58]. Also in patients who die suddenly of asthma, large numbers of neutrophils are found in their airways [59] although this may reect the rapid kinetics of neutrophil recruitment compared to eosinophil inammation. Rapid withdrawal of corticosteroids results in the appearance of neutrophils in the airways of patients with asthma [60]. There are data to suggest that monomeric IgE may mediate the appearance of neutrophils in the airways of asthmatics and may represent the anti-apoptic activity of myeloid cell leukemia-1 protein, decreased caspase-3 activity, diminished availability of Smac from mitochondria, and sequestration of the pro-apoptic protein Bax in the cytoplasm [61]. Thus the presence of neutrophils in the airway may reect the inammatory microenvironment rather than indicate a causal role for this cell. Further research into the role of neutrophils especially in severe asthma is ongoing [62].

T-lymphocytes
Th2 cells
T-lymphocytes play a very important role in co-ordinating the inammatory response in asthma through the release of specic patterns of cytokines, resulting in the recruitment and survival of eosinophils and in the maintenance of mast cells in the airways. T-lymphocytes are coded to express a distinctive pattern of cytokines, which are similar to that described

Pathophysiology of Asthma
Viruses Allergens
Allergen
Antigen presenting cell Dendritic cell, macrophage B7-2 CD28

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Dendritic cell Epithelial cells IL-17 TNF- CXCL1, CXCL8 IL-23

Th0
Allergenl peptide MHC1 TCR

TCR

Mast cell IL-12 IL-18

Th17
IL-4 IFN- T-bet Th17 IL-10 TGF- Th2 IL-4 IL-13 IL-5 IgE

Neutrophils Neutrophil elastase MMP-9 Goblet cells Fibroblast Mucus hypersecretion Fibrosis CXCL8 TGF-

Th1

IL-2 IFN-

Treg

Eosinophil

FIG. 33.2 Neutrophilic inflammation in asthma. Viruses, such as rhinovirus, stimulate the release of CXCL8 (IL-8) and CXCL1 (GRO-) from airway epithelial cells. Allergens activate dendritic cells to release IL-23, which recruits helper T-cells that secrete IL-17 (Th17 cells) to release tumor necrosis factor- (TNF-), which amplifies inflammation and CXCL1 and CXCL8, which recruit neutrophils into the airways. Neutrophils release more CXCL8 and also transforming growth factor- (TGF-), which activates fibroblasts to cause fibrosis, and neutrophil elastase and matrix metalloproteinase-9 (MMP-9) which stimulate mucus hypersecretion from goblet cells.

FIG. 33.3 T-lymphocytes in asthma. Asthmatic inflammation is characterized by a preponderance of T helper 2 (Th2) lymphocytes over T helper 1 (Th1) cells. Regulatory T-cells (Treg) have an inhibitory effect, whereas T helper 17 (Th17) cells have a proinflammatory effects. MHC1: Class 1 major histocompatibility complex; IL: interleukin; IFN-: interferon gamma; TGF-: transforming growth factor beta; IgE: immunoglobulin E, Th0: uncommitted T-cell.

in the murine Th2 type of T-lymphocytes, which characteristically express IL-4, IL-5, and IL-13 [6]. This programming of T-lymphocytes is presumably due to antigen-presenting cells such as dendritic cells, which may migrate from the epithelium to regional lymph nodes or which interact with lymphocytes resident in the airway mucosa. The naive immune system is skewed to express the Th2 phenotype; data now indicate that children with atopy are more likely to retain this skewed phenotype than are normal children [63]. There is also evidence that chitinase, a hydrolase that cleaves environmental chitin, an insect protein, plays a critical role in murine Th2 dierentiation and has been identied in human tissues from patients with asthma [64, 65]. The recruitment of Th2 cells in the airway is dependent on several chemotactic mediators, including the chemokines CCL17 (TARC) and CCL22 (MDC) which act on CCR4 that are selectively expressed on Th2 cells. These CCR4 ligands show increased expression in airway epithelial cells, macrophages, and mast cells of asthmatic patients [66]. PGD2, mainly derived from mast cells, is also an important chemoattractant of Th2 cells through the activation of DP2-receptors (or CRTH2) [67]. There is some evidence that steroid treatment may dierentially aect the balance between IL-12 expression and IL-13 expression [68]. Data from murine models of asthma [6971] have strongly suggested that IL-13 is both necessary and sufcient for induction of the asthmatic phenotype. One of the most important areas of asthma research in the next few years will be to establish the importance of IL-13 in the induction of the Th2 phenotype and asthma in humans.

Regulatory T-cells A subset of CD4 T-cells expressing CD25, termed T-regulatory cells (Tregs), are capable of suppressing both Th1 and Th2-mediated adaptive immune responses, and these cells may mediate their suppressive actions through release of TGF- or IL-10 [72]. An imbalance between allergen-specic Tregs and eector Th2 cells has been shown in allergic diseases [73]. There is some evidence that early infections or exposure to endotoxins might promote Th1mediated responses to predominate and that a lack of infection or a clean environment in childhood may favor Th2-cell expression and thus atopic diseases [74, 75]. Indeed, the balance between Th1 cells and Th2 cells is thought to be determined by locally released cytokines, such as IL-12, which tip the balance in favor of Th1 cells, or IL-4 or IL-13, which favor the emergence of Th2 cells (Fig. 33.3). Th17 cells Another subset of CD4 T-cells, known as Th17 cells, has recently been described and shown to have an important role in inammatory and autoimmune diseases [76]. Little is known about the role of Th17 cells in asthma, but increased concentrations of IL-17A (the predominant product of Th17 cells) have been reported in the sputum of asthma patients [77]. IL-17A and the closely related cytokine IL-17F have been linked to neutrophilic inammation by inducing the release of CXCL1 and CXCL8 from airway epithelial cells [78]. As well as IL-17, Th17 cells also produce IL-21, which is important for the dierentiation of these cells and thus acts as a positive autoregulatory mechanism, but it also
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Asthma and COPD: Basic Mechanisms and Clinical Management inhibits FOXP3 expression and regulatory T-cell development [79, 80]. Another cytokine IL-22 is also released by these cells and stimulates the production of IL-10 and acute-phase proteins [81]. However, more work is needed to understand the role and regulation of Th17 cells in asthma, as they may represent important new targets for future therapies.

Basophils
The role of basophils in asthma is uncertain, as these cells have previously been dicult to detect by immunocytochemistry [89]; indeed there may be multiple phenotypes of basophils with some able to release histamine on IgE challenge and others not able to do so [90]. Using a basophil-specic marker, a small increase in basophils has been documented in the airways of asthmatic patients, with an increased number after allergen challenge. However, these cells are far outnumbered by eosinophils [91].

Natural killer T-cells A subset of CD4 T-cells termed invariant natural killer T (iNKT) cells, which secrete IL-4 and IL-13, has been shown to account for 60% of all CD4 T-cells in bronchial biopsies from asthmatic patients [82], but this has been disputed in another study, which failed to show any increase in iNKT-cell numbers in bronchial biopsies, BAL or sputum of either asthma patients or COPD patients [83]. The role of iNKT cells in asthma is currently uncertain as there appears to be a discrepancy between the data from murine models of asthma and humans with the disease [84]. CD8 cells CD8 (cytotoxic) T-cells are present in patients with more severe disease and irreversible airow obstruction [85] and these cells may be of either the TC1 or the TC2 type, releasing the same patterns of cytokines as CD4 cells [86].

STRUCTURAL CELLS AND AIRWAY REMODELING

Structural cells of the airways, including epithelial cells, broblasts, and even airway smooth muscle cells are the site of inuence of many of the cytokines, growth factors, and inammatory mediators that characterize the asthmatic diathesis. Interestingly they may also be an important source of these same molecular entities [92, 93]. Although the concept is poorly dened, the idea that there are long-lasting changes in the cells that make up the airway wall and that these cells narrow the lumen and contribute to chronic airow limitation has been termed airway remodeling. Given the absence of a clean case denition, most investigators agree that certain changes, such as thickening of the collagen layer deposited below the true basement membrane, is a critical component of this aspect of asthma [9498]. Indeed, because structural cells far outnumber inammatory cells they may become the major source of mediators driving chronic inammation in asthmatic airways. For example, epithelial cells may have a key role in translating inhaled environmental signals into an airway inammatory response and are probably a target cell for inhaled glucocorticoids and anti-leukotrienes (Fig. 33.4) [99].

B-lymphocytes
B-cells have an important role in asthma through the release of allergen-specic IgE which binds to FcRI on mast cells and basophils and to low-anity FcRII on other inammatory cells, including B-cells, macrophages, and possibly eosinophils [87]. The Th2-type cytokines IL-4 and IL-13 induce B-cells to undergo class switching to produce IgE. In both atopic asthma and non-atopic asthma, IgE may be produced locally by B-cells in the airways [88].

Mechanical stress Viruses O2, NO2

Allergens

Viruses

FcRII

Macrophage TNF-, IL-1, IL-6

Airway epithelial cells GM-CSF CCL11 CCL5 CCL13 PDGF FGF IGF-1 IL-11 TGF ET Fibroblast activation

CCL5 IL-16 CCL17, CCL22

PDGF EGF

Eosinophil survival chemotaxis

Lymphocyte activation

Smooth muscle hyperplasia

FIG. 33.4 Airway epithelial cells in asthma. Airway epithelial cells may play an active role in asthmatic inflammation through the release of many inflammatory mediators, cytokines, chemokines and growth factors. TNF-: tumor necrosis factor alpha; IL: interleukin, CCL: C-C chemokine, PDGF: platelet-derived growth factor; EGF: epidermal growth factor; FGF: fibroblast growth factor; IGF: insulin-like growth factor.

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Pathophysiology of Asthma Structural changes in the airways may account for the accelerated decline in airway function seen in asthmatic patients over several years [100, 101]. Structural changes in asthmatic airways include

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increased thickness of airway smooth muscle; inltration of the airway smooth muscle with mast cells brosis (which is predominantly subepithelial); increased mucus-secreting cells; increased numbers of blood vessels (angiogenesis).

other inammatory cells. Because each mediator has many eects in the role of individual mediators in the pathophysiology of asthma is not yet clear. Although the multiplicity of mediators makes it unlikely that preventing the synthesis or action of a single mediator will have a major impact in clinical asthma, recent clinical studies with anti-leukotrienes suggest that cysteinyl leukotrienes have a clinically important eect.

Leukotrienes
The cysteinyl leukotrienes LTC4, LTD4, and LTE4 are potent constrictors of human airways and have been reported to increase AHR and may play an important role in asthma [107110] (see Chapter 24). The recent development of potent specic leukotriene antagonists has made it possible to evaluate the role of these mediators in asthma. Potent LTD4 antagonists protect (by about 50%) against exercise- and allergen-induced bronchoconstriction [111114], suggesting that leukotrienes contribute to bronchoconstrictor responses. Combined treatment with an anti-histamine and an antileukotriene is particularly eective [115, 116] Chronic treatment with anti-leukotrienes improves lung function and symptoms in asthmatic patients, although the degree of lung function improvement is not as great as that seen with an inhaled corticosteroid. It is only through the use of specic antagonists that the role of individual mediators of asthma may be dened. In the future, pharmaceuticals with specic targets of action will be of special value in providing pathobiological insights into the basic mechanisms of asthma; their potential role in the treatment of asthma remains to be determined.

It is not known if these changes are reversible with therapy. These changes may occur in some patients to a greater extent than others and may be increased by other factors such as concomitant cigarette smoking. It is likely that genetic factors will inuence the extent of remodeling that occurs in individual patients. There are good data to support the idea that mechanical deformation of the airway epithelium as a result of airway smooth muscle constriction results in the creation of a microenvironment that promotes airway remodeling. Deformed airway epithelial cells activate signaling via the epidermal growth factor receptor (EGFR) and the urokinase plasminogen activator system. Experimental data show that soluble signals generated by airway epithelial cells can inuence the phenotype of broblasts to produce collagen similar to that found in remodeled airways [102104]. These data could help explain why combination treatments with inhaled steroids and longacting -agonists are eective asthma therapies.

INFLAMMATORY MEDIATORS
Many dierent mediators have been implicated in asthma. They may have a variety of eects on the airways, which could account for the pathological features of asthma (Fig. 33.5) [105, 106]. Mediators such as histamine, prostaglandins, and leukotrienes contract airway smooth muscle, increase microvascular leakage, increase airway mucus secretion, and attract

Platelet-activating factor
Platelet-activating factor (PAF) and PAF-acetylhydrolase, the enzyme that degrades PAF, constitute a potent inammatory mediator system that mimics many of the features of asthma, including eosinophil recruitment and activation and induction of AHR; yet even potent PAF antagonists, such as modipafant, do not control asthma symptoms, at least in chronic asthma [117120]. However, genetic studies in Japan, where there is a high frequency of a genetic mutation which disables the PAF metabolizing enzyme, PAF-acetylhydrolase, have shown that there is an association between the presence of the mutant form of the enzyme and severe asthma [121, 122]. In addition, there are data showing that patients with a deciency of the enzyme that degrades PAF is associated with enhanced asthma severity [123]. These data suggest that there may be certain conditions associated with a signicant role for PAF in asthma.

Inflammatory cells Mast cells Eosinophils Th2 cells Basophils Neutrophils Platelets Structural cells Epithelial cells Sm muscle cells Endothelial cells Fibroblasts Nerves

Mediators Histamine Leukotrienes Prostanoids PAF Kinins Adenosine Endothelins Nitric oxide Cytokines Chemokines Growth factors

Effects Bronchospasm Plasma exudation Mucus secretion AHR Structural changes

Cytokines
Cytokines are increasingly recognized to be important in chronic inammation and play a critical role in orchestrating the type of inammatory response (see Chapter 27) (Fig. 33.6) [124]. Many inammatory cells (macrophages, mast cells, eosinophils, and lymphocytes) are capable of synthesizing
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FIG. 33.5 Multiple cells, mediators and effects. Many cells and mediators are involved in asthma and lead to several effects on the airways. Th2: T helper 2 cells; Sm: smooth; PAF: platelet-activating factor; AHR: airway hyperresponsiveness.

Asthma and COPD: Basic Mechanisms and Clinical Management

Allergen Epithelial cells IL-1 TNF- IL-12, IL-18 IL-10 CCL1 GM-CSF IL-3 IL-9 TNF- IL-4 IL-5 GM-CSF, IL-6, IL-11, IL-16 CCL11, CCL5, CXCL8, CCL17 Dendritic cell Smooth muscle Macrophage Th0 cell IL-4 IL-13 Th2 cell IL-4 IL-13 IL-5 GM-CSF CCL5, CCL11 FIG. 33.6 The cytokine network in asthma. Many inflammatory cytokines are released from inflammatory and structural cells in the airway and orchestrate and perpetuate the inflammatory response.

Monocyte

IL5, GM-CSI IgE B-Iymphocyte Eosinophil

Mast cell

and releasing these proteins, and structural cells such as epithelial cells and endothelial cells may also release a variety of cytokines and may therefore participate in the chronic inammatory response [125, 126]. There are number of major classes of cytokines. One class includes T-cell derived cytokines, examples of which include interferon (IFN) and TNF from Th1 cells and IL-4 and IL-5 from Th2 cells. Another class includes pro- and anti-inammatory cytokines with IL1 and IL-10 being respective examples. Chemoattractant cytokines, such as CCL11 and CXCL8 (IL-8), and growth factors cytokines, including PDGF, TGF-, and VEGF are other major classes. It is now clear that cytokines play a dominant role in chronic inammation of asthma. T-cell-derived cytokines play a key role in creating the microenvironment that lends itself to initiating and perpetuating the chronic inammatory response of asthma [127]. The key cytokines here are Th2 cytokines, IL-3, IL-4, and IL-5. IL-3 induces eosinophilia in vivo and maintains mast cell viability. IL-4 induces dierentiation of Th2 cells and creates the microenvironment in which IgE antibody is synthesized [128] and enhances mucin expression in goblet cells [129]. IL-5 enhances eosinophil maturation and prevents apoptosis of inammatory cells [130]. The recently described IL-17 family of cytokines, produced by cells as a result of the action of IL-15 or IL-23 on CD4 or CD8 memory cells may be important regulators of the asthmatic response [131133]. A lack of specic small molecule antagonists has made cytokine related research in humans dicult. Although important observations have been made using specic neutralizing antibodies [134], interventions involving immune function can be dicult. There is increased gene expression of IL-5 in lymphocytes in bronchial biopsies of patients with symptomatic asthma [135, 136]. The role of IL-5 in eosinophil recruitment in asthma has been conrmed in a study in which administration of an anti-IL-5 antibody to asthmatic

patients was associated with a decrease in eosinophil counts in the blood and BAL uid [49]. Interestingly in this study there was no eect on the physiology of the allergeninduced asthmatic response; although this is not the last word on eosinophils in asthma, it provides evidence that the eosinophil, as recruited by IL-5, is not the major pathogenetic cell in asthma. This has been conrmed by a study of the same antibody in symptomatic asthmatic patients, who showed no improvement in asthma control despite a profound reduction in circulating eosinophils [137]. Another Th2 cytokine produced by peripheral blood cells of patients with asthma, IL-9, may play a critical role in airway remodeling, eosinophilopoiesis, and may interact with the IL-4 receptor in mediating some of the pathobiological features of asthma [138141]. Other cytokines, such as IL-1, IL-6, TNF-, TGF, and GM-CSF, are released from a variety of cells, including macrophages and epithelial cells and may be important in amplifying the inammatory response. TNF- may be an amplifying mediator in asthma and is produced in increased amounts in asthmatic airways. Inhalation of TNF- increased airway responsiveness in normal individuals [142]. TNF- and IL-1 both activate the proinammatory transcription factors, nuclear factor-B (NF-B) and activator protein-1 (AP-1) which then switch on many inammatory genes in the asthmatic airway [143]. TGF-, through ligation of two receptors, type I and II, has been linked to tissue remodeling, and potentially to subepithelial brosis through the Smadsignaling pathway which could be a site for therapeutic intervention [144146]. Thymic stromal lymphopoeitin (TSLP) is a cytokine that shows a marked increase in expression in airway epithelium and mast cells of asthmatic patients [66]. TSLP appears to play a key role in programming airway dendritic cells to release CCL17 and CCL22 to attract Th2 cells (Fig. 33.7) [147]. It therefore acts as an upstream cytokine which

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Pathophysiology of Asthma peroxidation of their lipid membranes. Phagocytes use heme peroxidases (myeloperoxidase, MPO) or eosinophil peroxidase (EPO) to produce ROS; these moieties can also interact with nitrite (NO2) and hydrogen peroxide (H2O2) to promote formation of RNS. Eosinophils, a key cell in asthma, have several times greater capacity to generate O2 and H2O2 than do neutrophils, and the content of EPO in eosinophils is 310 times higher than the amount of MPO present in neutrophils [153155]. Evidence for increased oxidative stress in asthma is provided by the increased concentrations of 8-isoprostane (a product of oxidized arachidonic acid) in exhaled breath condensates [156], and increased ethane (a product of oxidative lipoid peroxidation) in exhaled breath of asthmatic patients [156158]. Airow obstruction, airway hyperresponsiveness and airway remodeling have been associated with impaired SOD activity [159, 160]. Oxidative stress is increased particularly in patients with severe asthma (perhaps as a result of the neutrophilic inltration in these patients) and this may reduce the activity of histone deacetylase (HDAC), which may account for the reduced response to corticosteroids in these patients [161, 162].

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Epithelial cells

Mast cell
Y

TSLP Myeloid dendritic cell CCL17 CCR4 Th2 cell IL-5 IL-4, IL-13 IgE Eosinophil B-lymphocyte

FIG. 33.7 Thymic stromal lymphopoetin in asthma. TSLP is an upstream cytokine produced by airway epithelial cells and mast cells in asthma that acts on immature dendritic cells to mature and release CCL17 (TARC), which attracts Th2 cells via CCR4. Th2: T helper 2; CCL: chemokine; CCR: chemokine receptor.

Endothelins
Endothelins are potent peptide mediators that are vasoconstrictors, bronchoconstrictors and promote brosis [163, 164]. Endothelin-1 levels are increased in the sputum, serum, and BAL uid of patients with asthma; these levels are modulated by allergen exposure and steroid treatment [165167]. Endothelins also induce airway smooth muscle cell proliferation and promote a probrotic phenotype and may therefore play a role in the chronic inammation of asthma [164, 168].

may play a key role in orchestrating an allergic pattern of inammation in asthmatic airways.

Complement
A decade ago it was postulated that complement had little role in the biology of asthma. However, studies in mice harboring a targeted deletion of the C5a receptor showed diminished bronchial hyperresponsiveness induced after allergen challenge [148]. In addition, genetic linkage studies in mice have linked regions of the mouse genome containing the gene for C5a to the phenotype of AHR [149]. Furthermore, the complement peptide C5a has been recovered from BAL uid of patients after allergen challenge [148]. Current thinking is that complement contributes to the airway inammation, AHR and mucus production observed in human asthma and may also prevent the formation of a Th2-directed immune response to allergens [150152].

Nitric oxide
Nitric oxide (NO) is produced by several cells in the airway by NO synthases [169, 170] (see Chapter 30). Although the cellular source of NO within the lung is not known, inferences based on mathematical models suggest that it is the large airways which are the source of NO [171]. However, in patients with severe asthma NO is also derived form peripheral airways [172]. Current data indicate that the level of NO in the exhaled air of patients with asthma is higher than the level of NO in the exhaled air of normal subjects (see Chapter 30) [173177]. The elevated levels of NO in asthma are closely linked to airway pH [178180] and thus are more likely reective of inammatory mechanisms than of a direct pathogenetic role of this gas in asthma [181, 182]. The combination of increased oxidative stress and NO may lead to the formation of the potent radical peroxynitrite that may result in nitrosylation of proteins in the airways [183, 184]. Asthmatics managed by measurements of exhaled nitric oxide achieved equivalent asthma control with less inhaled corticosteroids than those managed using conventional algorithms [185]; the availability of portable devices to measure exhaled nitric oxide may allow management of asthma in manner similar to the self management of diabetics [186].

Oxidative stress
In asthma, activation of epithelial cells, and resident macrophages, and the recruitment and activation of neutrophils, eosinophils, monocytes, and lymphocytes leads to the generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS). Action of many systems but primarily NADPH oxidase results in the microenvironmental availability of superoxide anions (O2), which are rapidly converted to H2O2 through the catalytic action of superoxide dismutase (SOD). Hydroxyl ion (OH) is then formed nonenzymatically; this damages tissues, particularly cell membranes by

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EFFECTS OF INFLAMMATION ON THE AIRWAYS

The idea that the chronic inammatory response results in the characteristic pathophysiological changes associated with asthma has been widely promulgated. However, biopsies from children with wheezing show many of the characteristic features of asthma [12]. Thus it is probable that some of the dierences between the condition of the airways in normal and asthmatic subjects reects the genetic makeup of the patient inuenced by environmental factors such as airway infections and allergen exposures. The descriptions below present the observations without adequate evidence to support whether changes are markers for or the result of a chronic inammatory response. The inammatory response has many eects on structural cells of the airways resulting an amplication and perpetuation of the inammatory response (Fig. 33.8).

inammatory cells, and activated complement. Epithelial cells shed from the airway wall may be found in clumps in the BAL uid or sputum (Creola bodies) of asthmatics, suggesting that there has been a loss of attachment to the basal layer or basement membrane. The damaged epithelium may contribute to AHR in a number of ways, including

loss of its barrier function to allow penetration of allergens and irritants; loss of enzymes (such as neutral endopeptidase) which normally degrade inammatory mediators such as kinins; loss of a relaxant factor (so-called epithelial-derived relaxant factor); edema and swelling that promotes obstruction by lumenal encroachment; loss of surfactant function leading to increased increased recoil of the airway; exposure of sensory nerves which may lead to reex neural eects on the airway.

Airway epithelium Fibrosis

Airway epithelial cells play a critical role in asthma and link environmental inuences to inammatory responses in the airway (Fig. 33.4). Airway epithelial shedding may be important in contributing to AHR and may explain how several dierent mechanisms, such as ozone exposure, certain virus infections, chemical sensitizers and allergen exposure can lead to its development, since all these stimuli may lead to epithelial disruption. Epithelium may be shed because of an inammatory milieu made up of eosinophil basic proteins, oxygen-derived free radicals, proteases released from The basement membrane in asthma appears on light microscopy to be thickened; on closer inspection by electron microscopy it has been demonstrated that this apparent thickening is due to subepithelial brosis with deposition of types III and V collagen below the true basement membrane; however, the ratio of interstitial collagen brils to matrix is not different from that found in normal subjects [187189]. Data from a number of investigative groups show that the thickness of the deposited collagen is related to airway obstruction

Allergen

Macrophage/ dendritic cell Th2 cell

Mast cell

Neutrophil Eosinophil Mucus plug Nerve activation Subepithelial fibrosis Sensory nerve activation Cholinergic reflex Bronchoconstriction Hypertrophy/hyperplasia Epithelial compression and loss

Plasma leak Edema Mucus hypersecretion hyperplasia Vasodilatation New vessels

Myofibroblast

FIG. 33.8 The complexity of asthma. The pathophysiology of asthma is complex, with participation of several interacting inflammatory cells which result in acute and chronic inflammatory effects on the airway.

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Pathophysiology of Asthma and airway responsiveness [190193]. The mechanism of the collagen deposition is not known. However, it is known that several probrotic cytokines, including TGF- and PDGF, and mediators such as endothelin-1, can be produced by epithelial cells or macrophages in the inamed airway [126]. Even simple mechanical manipulations can alter the phenotype of airway epithelial cells in a probrotic fashion [102104, 194]. The role of airway brosis in asthma is unclear, as subepithelial brosis has been observed even in mild asthmatics at the onset of disease; it is not certain whether the collagen deposition has any functional consequences. Subepitheial brosis is also seen in patients with chronic eosinophilic bronchitis, who usually present with cough and have an inltration of eosinophils in the airways but no AHR or airway obstruction [195]. This suggests that the subepithelial brosis seen in asthmatics is likely to be secondary to eosinophilic inammation in the airways and may not have important functional consequences. smooth muscle modulates the remodeling process by secreting multiple cytokines, growth factors, or matrix proteins and by expressing cell adhesion molecules and other potential costimulatory molecules [196]. For example, increased numbers of mast cells have been identied in airway smooth muscle of patients with asthma [15, 198]. The importance of airway smooth muscle in chronic asthma was highlighted by a recent trial in which airway smooth muscle was rendered dysfunctional by bronchial thermoplasty. Although this intervention was limited to relatively large airways there was a measurable, although not overwhelming, clinical benet [203]. The idea of manipulating the mass or contractile capacity of airway smooth muscle in asthma remains an important and unexplored avenue of asthma therapeutics [199, 204].

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Vascular responses
It has been known for decades that the subepithelial connective tissue of the asthmatic airway has many more blood vessels than are found in similar locations in normal subjects [205]. It is now recognized that bronchial vessels play a key role in the pathophysiology of asthma (Fig. 33.10). Despite this anatomic knowledge, little is known about the role of the bronchial circulation in asthma. This is partly because of the diculties involved in measuring airway blood ow. Recent studies using an inhaled absorbable gas have demonstrated increased airway mucosal blood ow in asthma [206208]. The bronchial circulation may also play an important role in regulating airway caliber, since an increase in the vascular volume may contribute to airway narrowing. Vascular blanching of the skin is used to measure the potency of topical steroids and a similar mechanism may be involved with the eects of inhaled steroids in asthma [207, 209]. Increased airway blood ow may be important in removing inammatory mediators from the airway, and may play a role in the development of exercise-induced asthma [210]. Increased shear stress due to high expiratory pressures may lead to gene transduction and enhanced production of nitric oxide by type III (endothelial) NO synthase [211, 212]; there are in vivo experimental data in sheep that support the importance of this mechanism [213]. There may also be an

Airway smooth muscle

When the concept that asthma was primarily an inammatory disease was developed in the late 1980s to early 1990s, airway smooth muscle was thought to be predominantly a bystander tissue. That is the inammatory process resulted in the availability of mediators that led to smooth muscle constriction and expression of an asthma phenotype. There are now substantial data to support the concept that airway smooth muscle in asthma is phenotypically dierent from normal airway smooth muscle and that these changes reect the impact of the inammatory asthmatic airway microenvironment on smooth muscle [15, 196201]. Although some of the abnormalities identied to date in asthma impact on the contractility of individual units of airway smooth muscle, the more recently identied ones reect dierences in the ability of this tissue to proliferate or to function as an endocrine tissue (Fig. 33.9). For example, airway smooth muscle may increase in mass because of an increased tendency to proliferate due to the absence of an inhibitory transcription factor C/EBP- [197]. Airway smooth muscle is not only inuenced by the inammatory mediators and cytokines in which it is immersed [202], but also recent studies suggest that airway
Inflammatory cells

Mediators Airway smooth muscle

Contraction
Histamine, cys -leukotrienes kinins, prostanoids, endothelin

Proliferation
PDGF, EGF, endothelin-1

Secretion
cytokines, chemokines, prostanoids

FIG. 33.9 Airway smooth muscle in asthma. Inflammation has several effects on airway smooth muscle cells, resulting in contraction, proliferation and secretion of inflammatory mediators. Cys: Cysteinyl; PDGF: platelet-derived growth factor; EGF: epidermal growth factor.

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Asthma and COPD: Basic Mechanisms and Clinical Management increase in the number of blood vessels in asthmatic airways as a result of angiogenesis in response to VEGF [214216]. Microvascular leakage is an essential component of the inammatory response and many of the inammatory mediators implicated in asthma produce this leakage [217, 218]. There is a good evidence for microvascular leakage in asthma, and it may have several consequences on airway function, including increased airway secretions, impaired mucociliary clearance, formation of new mediators from plasma precursors (such as kinins), and mucosal edema which may contribute to airway narrowing and increased AHR [219221].

Mucus hypersecretion
Mucus hypersecretion is a common inammatory response in secretory tissues and is an important component of the inammatory response in asthma (Fig. 33.11). Increased mucus secretion contributes to the viscid mucus plugs which occlude asthmatic airways, particularly in fatal asthma. There is evidence for hyperplasia of submucosal glands which are conned to large airways, and of increased numbers of epithelial goblet cells in patients with asthma, especially those with severe asthma [8]. One of the major mucin genes, MUC5AC is induced by complement factor C3a [222], NO [223], TNF- [224], EGF family ligands [225], or neutrophil elastase [226]. Since both neutrophils and enhanced expression of mucin genes have been found in great profusion in the airways of patients with severe asthma [52], it is tempting to speculate that the neutrophils may have led to the enhanced mucin gene expression. Thus enhanced mucin expression in epithelial cells can be viewed as an indicator of the inammatory state of the airways. These ndings are not limited to cell culture, since in intact humans with asthma, exercise-induced bronchospasm is associated with enhanced expression of MUC5AC [227]. It is highly likely that methods to manipulate mucin gene expression in response to inammatory stimuli will be identied and could be of value in the treatment of asthma as well as COPD.

Inflammatory mediators

Vasodilatation
NO, CGRP histamine, PGE2

Plasma exudation
histamine, cys -leukotrienes kinins, PAF

Angiogenesis
VEGF, TNF-

FIG. 33.10 Vascular abnormalities in asthma. Allergic inflammation has several vascular effects, including vasodilatation, plasma exudation from post-capillary venules and new vessel formation (angiogenesis). NO: Nitric oxide; CGRP: calcitonin gene-related peptide; PGE2: prostaglandin E2; PAF: platelet-activating factor; VEGF: vascular-endothelial growth factor; TNF-: tumor necrosis factor-alpha.

Neural effects
Neural mechanisms play an important role in the pathogenesis in asthma as discussed in Chapter 32 [228]. Autonomic nervous control of the airways is complex; in addition to classical cholinergic and adrenergic mechanisms,

Neutrophil

Th2 cell Oxidative stress IL-4, IL-13, IL-9

Neutrophil elastase EGFR

MUC5AC

Goblet cells

Goblet cells Mucus hypersecretion Submucosal gland

Mucus hyperplasia

FIG. 33.11 Mucus hypersecretion in asthma. Increased mucus secretion in asthma may be stimulated by neutrophils through the release of neutrophil elastase, T helper 2 (Th2) cytokines and oxidative stress. This may be mediated via epidermal growth factor receptors (EGFR) which may result in mucus hyperplasia and increased expression of the mucin gene MUC5AC. Mucus hypersecretion is also enhanced by neural mechanisms through the release of acetylcholine (ACh) and substance P (SP).

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Pathophysiology of Asthma nonadrenergic noncholinergic (NANC) nerves and several neuropeptides have been identied in the respiratory tract [229231]. Many studies have investigated the possibility that defects in autonomic control may contribute to AHR in asthma, and abnormalities of autonomic function, such as enhanced cholinergic and -adrenergic responses or reduced -adrenergic responses, have been proposed. Current thinking suggests that these abnormalities are likely to be secondary to the disease, rather than primary defects [228]. It is possible that airway inammation may interact with autonomic control by several mechanisms. There is a close interaction between airway inammation and airway nerves (Fig. 33.12). Inammatory mediators may act on various prejunctional receptors on airway nerves to modulate the release of neurotransmitters [232, 233]. Thus thromboxane and PGD2 facilitate the release of acetylcholine from cholinergic nerves in canine airways, whereas histamine inhibits cholinergic neurotransmission at both parasympathetic ganglia and postganglionic nerves via histamine H3-receptors. Inammatory mediators may also activate sensory nerves, resulting in reex cholinergic bronchoconstriction or release of inammatory neuropeptides. Inammatory products may also sensitize sensory nerve endings in the airway epithelium, so that the nerves become hyperalgesic. Hyperalgesia and pain (dolor) are cardinal signs of inammation, and in the asthmatic airway may mediate cough and chest tightness, which are such characteristic symptoms of asthma. The precise mechanisms of hyperalgesia are not yet certain, but mediators such as prostaglandins, certain cytokines, and neurotrophins may be important. Neurotrophins, which may be released from various cell types in peripheral tissues, may cause proliferation and sensitization of airway sensory nerves [234]. Bronchodilator nerves which are nonadrenergic are prominent in human airways and may be dysfunctional in asthma [235]. In human airways, the bronchodilator neurotransmitter appears to be NO [236]. Airway nerves may also release neurotransmitters which have proinammatory eects. Thus neuropeptides such as substance P (SP), neurokinin A, and calcitonin-gene related peptide may be released from sensitized inammatory nerves in the airways which increase and extend the ongoing inammatory response [237]. There is evidence for an increase in SP-immunoreactive nerves in airways of patients with severe asthma [238], which may be due to proliferation of sensory nerves and increased synthesis of sensory neuropeptides as a result of nerve growth factors released during chronic inammation although this has not been conrmed in milder asthmatic patients [239]. There may also be a reduction in the activity of enzymes, such as neutral endopeptidase, which degrade neuropeptides such as SP [240]. Thus chronic asthma may be associated with increased neurogenic inammation, which may provide a mechanism for perpetuating the inammatory response even in the absence of initiating inammatory stimuli.

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TRANSCRIPTION FACTORS
The chronic inammation of asthma is due to increased expression of multiple inammatory proteins (cytokines, enzymes, receptors, adhesion molecules). In many cases these inammatory proteins are induced by transcription factors, DNA-binding factors that increase the transcription of selected target genes (Fig. 33.13) [143]. A proinammatory transcription factor that may play a critical role in asthma is nuclear factor-kappa B (NF-B) which can be activated by multiple stimuli, including protein kinase C activators, oxidants, and proinammatory cytokines (such as IL-1 and TNF-) [241243]. There is evidence for increased activation of NF-B in asthmatic airways, particularly in epithelial cells and macrophages [244]. Although many aspects of the asthmatic airway are inuenced by the expression of transcription factors, there are data linking the mechanical deformation of the airway to expression of proinammatory transcription factors; these ndings have direct implications for the mechanism of exercise-induced asthma and may explain how this physical stimulus could

Inflammatory mediators Neutrotrophins

Airway nerves Inflammatory cells

Neurotransmitters Neuropeptides Neurogenic inflammation FIG. 33.12 Neural mechanism in asthma. There is a close interaction between asthmatic inflammation and neural mechanisms.

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Asthma and COPD: Basic Mechanisms and Clinical Management

Inflammatory stimuli
allergens, viruses, cytokines

Inflammatory proteins
cytokines, enzymes, receptors, adhesion molecules

Receptor

Kinases

Inactive transcription factor mRNA Activated transcription factor (NF-B, AP-1, STATs)

P Nucleus

Inflammatory gene

Promoter

Coding region

FIG. 33.13 Proinflammatory transcription factors in asthma. Transcription factors play a key role in amplifying and perpetuating the inflammatory response in asthma. Transcription factors, including nuclear factor kappa-B (NF-B), activator protein-1 (AP-1), and signal transduction-activated transcription factors (STATs), are activated by inflammatory stimuli and increase the expression of multiple inflammatory genes.

IL-27

IL-12

IL-4

IL-33

STAT1

STAT4

STAT6

Th1 cells

T-bet

GATA-3

Th2 cells

Th1 cytokines (IL-2, IFN-)

Th2 cytokines (IL-4, IL-5, IL-9, IL-13) Allergic inflammation

FIG. 33.14 GATA-3 in asthma. The transcription factor GATA-3 (GATAbinding protein-3) is regulated by interleukin-4 (IL-4) via STAT-6 (signal transducer and activator of transcription 6) and regulates the expression of IL-4, IL-5, IL-9, and IL-13 from T helper 2 (Th2) cells and inhibits the expression of T-bet via inhibition of STAT4. IL-33 enhances the actions of GATA-3. T-bet regulates TH1-cell secretion of IL-2 and interferon- (IFN-) and has an inhibitory action on GATA-3. T-bet is regulated by IL-12 via STAT4 and by IL-27 via STAT1. This demonstrates the complex interplay of cytokines and transcription factors in asthma.

modify the inammatory microenvironment of the airway [104, 245247]. The transcription factor GATA-3 (GATA-binding protein 3) is crucial for the dierentiation of uncommitted
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naive T-cells into Th2 cells and it regulates the secretion of TH2-type cytokines [248]. There is an increase in the number of GATA-3 T-cells in the airways of asthmatic subjects compared with normal subjects [249]. Following simultaneous ligation of the T-cell receptor (TCR) and co-receptor CD28 by antigen-presenting cells, GATA-3 is phosphorylated and activated by p38 mitogen-activated protein kinase. Activated GATA-3 then translocates from the cytoplasm to the nucleus, where it activates gene transcription [250]. GATA-3 expression in T-cells is regulated by the transcription factor STAT-6 (signal transducer and activator of transcription 6) via IL-4 receptor activation. Another transcription factor T-bet has the opposite eect and promotes Th1 cells and suppresses Th2 cells. T-bet expression is reduced in T-cells from the airways of asthmatic patients compared with nonasthmatic patients [251]. When phosphorylated, T-bet can associate with and inhibit the function of GATA-3, by preventing it from binding to its DNA target sequences [252]. GATA3 inhibits the production of Th1-type cytokines by inhibiting STAT-4, the major transcription factor activated by the T-bet-inducing cytokine IL-12 [253] (Fig. 33.14). Nuclear factor of activated T-cells (NFAT) is a T-cell-specic transcription factor and appears to enhance the transcriptional activation of GATA-3 at the IL-4 promoter [254]. FOXP3 is a transcription factor that is expressed in Tregs, whereas the equivalent transcription factor in Th17 cells is RORt. There appears to be a negative interaction between these transcription factors so that a reduction of Tregs in asthma may be a factor increasing Th17 cells [255].

Pathophysiology of Asthma exhaled breath condensates from healthy subjects but levels were signicantly lower in condensates from patients with asthma exacerbations [266]. PD1 when administered before inhaled allergen challenge, in mice led to decreased airway eosinophil and T-lymphocyte recruitment, decreased airway mucus, and less of an increment in AHR than without the PD1. These data suggest that endogenous PD1 is a novel and important counterregulatory signal in allergic airway inammation.

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Antiinflammatory mediators
IL-10, IL-1ra IFN-, IL-12, IL-18 Lipoxins, resolvins Protectins PGE2

Inflammatory mediators
Lipid mediators Cytokines Peptides Oxidants

IL-10
Airway and alveolar macrophages have a predominantly suppressive eect in asthma and inhibit T-cell proliferation. The mechanism of macrophage-induced immunosuppression is not yet certain, but PGE2 and IL-10 secretion may contribute [267]. IL-10 inhibits many of the inammatory mechanisms in asthma and its secretion is defective from macrophages of asthmatic patients [268]. IL-10 is secreted by a subset of Tregs and this is enhanced by specic allergen immunotherapy [267]. Interestingly treatment of asthmatic children with an inhaled corticosteroid or with montelukast increases serum levels of IL-10 [269]; these ndings suggest that increasing IL-10 may be an important therapeutic approach in asthma.

FIG. 33.15 Anti-inflammatory mediators in asthma. There may be an imbalance between increased proinflammatory mediators and a deficiency in anti-inflammatory mediators. IL: interleukin; IL-1ra: interleukin-1 receptor antagonist; IFN-: interferon gamma; PGE2: prostaglandin E2.

ANTI-INFLAMMATORY MECHANISMS IN ASTHMA

Although most emphasis has been placed on inammatory mechanisms, there may be important anti-inammatory mechanisms that may be defective in asthma, resulting in increased inammatory responses in the airways (Fig. 33.15).

GENETIC INFLUENCES
Over the past 15 years there have been multiple studies on the genetics of asthma (see Chapter 4). A number of potential asthma genes have been identied [270], but very few have been replicated in multiple populations [271]. However all the data suggest that there is not a single major asthma susceptibility gene, but rather there are multiple genes each explaining only a small proportion of the total variance in the asthma phenotype. The keys to nd the most important asthma genes will be to use comprehensive methods and to replicate the ndings in an individual population in multiple distinct populations. ADAM33 is an asthma gene, discovered by positional cloning, that has been replicated in many but not all populations tested [272]. It was initially identied in Caucasian families from the United States and United Kingdom and was associated with asthma characterized by bronchial hyperresponsiveness. There were many variants within ADAM33 that were signicantly associated with these phenotypes. Subsequently, there have been a large number of case-control and family-based association studies focused on ADAM33, with the majority but not all, conrming the original nding [273]. Current data suggest that ADAM33 may be involved in lung development and remodeling. In genome wide-association studies microarrays capable of typing 500,000 to 1 million single nucleotide polymorphisms are used to identify associations between a given phenotype and genotype. This technology has been applied to childhood asthma [274]. These investigators identied a strong statistical association between the diagnosis of
415

Lipid anti-inflammatory mediators

Several lipid mediators appear to have anti-inammatory eects, including PGE2, lipoxins, resolvins, and protectins [256, 257]. There is a strong evidence supporting the role of signaling through the lipoxin A receptor as being decient in severe asthma in general and aspirin-sensitive asthma in particular [258263]. In addition it has now been established that presqualene diphosphate (PSDP) is an endogenous regulator of phosphatidylinositol 3-kinase (PI3K). PSDP, but not presqualene monophosphate (PSMP), directly inhibits recombinant human PI3K- activity and a new PSDP structural mimetic blocks human neutrophil activation and murine lung PI3K activity and inammation [264]. Resolvin E1 (RvE1: 5S,12R,18R-trihydroxyei cosapentaenoic acid) is an anti-inammatory lipid mediator derived from the -3 fatty acid eicosapentaenoic acid. RvE1 is involved in the resolution of inammation and reduces allergic inammation in mice exposed to inhaled allergen [265]. Another important anti-inammatory mediator is protectin D1, a natural lipid based chemical mediator that counters leukocyte activation. PD1 has been recovered from

Asthma and COPD: Basic Mechanisms and Clinical Management asthma in childhood in families from the United Kingdom and markers located on chromosome 17q21. The results were replicated in two European populations. They then used the genetic data to examine transcript levels in immortalized lymphocytes from children harboring the implicated variants and showed a strong association between variants in ORMDL3 and asthma. ORMDL3 is a member of a family of genes that encode endoplasmic reticulum proteins of unknown function. If these studies are replicated, then the community of asthma researchers will need to determine how variants in the function of this gene could lead to childhood asthma. An area of importance is the relationship between clinical responses to anti-asthma treatments and genotype at various drug targets [275]. For example, there are known to be a number of SNPs in the 2-adrenoceptor gene that result in structural changes in the 2-receptor structure associated with functional changes in isolated cell systems [276]. These are related to the acute response to bronchodilator aerosols acting via this receptor [277, 278] and with deleterious eects of chronic use of albuterol [279, 280]. In a double blind randomized controlled trial, the rst in a nonmalignant condition where genotype at a specic locus was an entry criterion, those patients harboring the alleles encoding for homozygous expression of arginine rather than glycine at the 16th amino acid in the 2-adrenergic receptor had diminished morning and evening peak ow and lower FEV1 after the regularly scheduled use of albuterol than when albuterol was used on an as needed only basis [281]. In a retrospective analysis of two other randomized clinical trials similar changes have been observed by some groups [282] but not by others [283]. However, other retrospective analyses of long-acting 2-agonists have not demonstrated a functional eect of these SNPs [284]. Similarly, there are genetically based functional variants in the regulation of the ALOX-5 gene [285]. Although these variants were originally associated with the response to anti-leukotriene treatment [286], current studies suggest that it is not these variants per se, but variants in the ALOX-5 gene in general that may confer dierences in treatment response [287]. central heating providing more favorable environment), cigarette smoking (pregnancy and early childhood exposure), and possibly air pollution due to road trac. The role of exposures to pets in early childhood remains unresolved and because of confounding will be dicult to ascertain using an epidemiological approach. Why does asthma once established become chronic in some individuals but remain intermittent and episodic in others? Is there a genetic dierence among people or an environmental dierence among stimuli that results in full resolution of airway obstruction in some patients and the failure for this to occur in others? For example, occupational asthma due to chemical sensitizers, such as toluene diisocyanate, may remit if the patient is removed from exposure to the sensitizer within 6 months of development of asthma symptoms, whereas longer exposure is often associated with persistent asthma even when avoidance of exposure is complete [289, 290]. This suggests that once inammation is established it may continue independently of a causal mechanism, whereas in others this self perpetuation does not occur. Are there dierent types of asthma, each characterized by a common genetic background with its attendant immunological and inammatory response? Asthma remains a syndromic diagnosis. How can we get beyond a physiological and descriptive denition to one in which the primary disease pathobiology, for each of the many asthma syndromes, can be developed? How does inammation of the airways translate into clinical symptoms of asthma? Airway thickening, as a consequence of the inammatory response, may contribute to increased responsiveness to spasmogens [193, 291]. However, there is no obvious relationship between the inammatory response in airways and asthma severity; patients with mild asthma may have a similar eosinophil response to patients with severe asthma, suggesting that there are other factors that determine clinical severity. The nature of these factors remains elusive, but we must identify them if we are to make true scientic progress. How important are genetic factors (genetic polymorphisms) in determining the phenotype of asthma? Asthma is a complex genetic disease but likely one in which there is both dierences among individuals in the genes that pre-dispose to asthma and a requirement for more than one genetic variant in a given person for asthma to be manifest. Since each asthma gene may account for such a small proportion of the total genetic variance in asthma, how can we use genetics to develop better asthma diagnostics? How can we use genetics to help stratify patients to achieve uniform cohorts for clinical trials? Are asthma and atopy causally related or just closely linked? Much of our understanding of asthma derives from study of models of allergy. Although these models are of great value in dening the pathobiology of asthma, how closely will they relate to the human disease?

UNANSWERED QUESTIONS
As a syndrome much about asthma remains an enigma. Although we have reasonably eective treatments and a general idea of it pathophysiology, there are still many unanswered questions about its pathobiology. Why is the prevalence of asthma increasing throughout the world as a consequence of Westernization? It is clear that allergic diseases appear as societies become more Westernized, but what environmental factors are responsible is not known. It is likely that several factors are operating together [288]. Among the factors identied to date are diet (reduced intake of antioxidants, reduced unsaturated fats), lack of early childhood infections (with consequent tendency to develop Th2-driven responses), greater exposure to allergens in the home (tight housing, mattresses,

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33

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