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Zuyderduyn, S. (2007, November 27). Airway smooth muscle cells : regulators of airway inflammation. Retrieved from
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AIRWAY SMOOTH
MUSCLE CELLS:
regulators of
airway inflammation
Suzanne Zuyderduyn
regulators of airway inflammation
Proefschrift
ter verkrijging van
de graad van Doctor aan de Universiteit Leiden, op gezag van Rector Magnificus prof.mr. P.F. van der Heijden,
volgens besluit van het College voor Promoties te verdedigen op dinsdag 27 november 2007
klokke 13.45 uur
door
Suzanne Zuyderduyn geboren te Leiderdorp in 1975
Promotores Prof. Dr. P.S. Hiemstra Prof. Dr. K.F. Rabe
Referent Dr. S.J. Hirst (King’s College, Londen)
Overige leden Prof. Dr. P. ten Dijke Dr. C. van Kooten Prof. Dr. A. van der Laarse
Prof. Dr. H. Meurs (Universiteit Groningen)
The research described in this thesis was performed at the Department of Pulmonology of the Leiden University Medical Center and was supported by grants from AstraZeneca (Lund, Sweden) and the Netherlands Asthma Foundation (AF00.17)
Financial support for printing of this thesis was provided by:
Stichting Astmabestrijding Nederlands Astma Fonds
Cover and lay-out: Esther van den Oever, het Clickt Printed by: Drukkerij Lemver
ISBN nummer: 978-90-9022357-5
Chapter 1 General introduction and scope of the thesis
Chapter 2 Treating asthma means treating airway smooth muscle (ASM) cells
Submitted for publication
Chapter 3 TGF-β differentially regulates Th2 cytokine-induced eotaxin and eotaxin-3 release
Journal of Allergy and Clinical Immunology 2004; 114:791-798
Chapter 4 The antimicrobial peptide LL-37 enhances IL-8 release by human airway smooth muscle cells
Journal of Allergy and Clinical Immunology 2006; 117:1328-1335
Chapter 5 Epithelial differentiation is a determinant in the production of eotaxin-2 and -3 by bronchial epithelial cells in response to IL-4 and IL-13
Molecular Immunology 2007; 44:803-811
Chapter 6 Budesonide but not formoterol inhibits IL-4-induced eotaxin release by human airway smooth muscle cells
Submitted for publication
Chapter 7 General discussion
Chapter 8 Nederlandse samenvatting
Dankwoord Curriculum Vitae Publicaties
Supplement Full colour images
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127 147 155 157 159 161
CHAPTER 1
General introduction and scope of the thesis
ASTHMA
Asthma is defined as a chronic inflammatory disease characterized by (i) an exaggerated contractile response to various stimuli that do not provoke such an effect in healthy individuals (airway hyperresponsiveness, AHR) and (ii) variable airway obstruction caused by contrac- tion of the airway smooth muscle (ASM) layer, swelling of the airway wall and mucus hyper- secretion due to inflammation.
The pathogenesis of allergic asthma is complicated and consists of both host factors (atopy) and environmental factors (pollutants, viruses). Currently it is thought that T lymphocytes, as orchestrators of the inflammatory response, play a pivotal role in the pathogenesis of asthma (1). In asthmatics the T cells in bronchoalveolar lavage fluid and bronchial biopsies express cytokines consistent with a Th2 phenotype (2;3). Why the balance between Th1 and Th2 cells is altered towards the Th2 cells is unknown, but may be due to altered function of dendritic cells (DC) (4).Th2 lymphocytes in the airways express cytokines including IL-4, IL-5, IL-9 and IL-13, which can induce recruitment of eosinophils to the airways and affect function of epithelium and ASM cells (see Figure 1).The infiltrated eosinophils also affect epithelial and ASM function by releasing
their inflammatory mediators (e.g. eosinophil cationic protein [ECP], major basic protein [MBP], leukotrienes) and epithelial damage and AHR are the result.
9
Chapter 1
Figure 1.Effects of inflammatory cells on airway resident cells in asthma. Eosinophils release ECP and MBP which can induce epithelial damage; these products, leukotrienes and TNF-α induce AHR.
Mast cell products (e.g. histamine and tryptase) induce AHR. Cytokines released by Th2 lymphocytes (IL-4, IL-5, IL-9 and IL-13) induce goblet cell hyperplasia. Abbreviations: ASM, airway smooth muscle;
ECP, eosinophilic cationic protein; eos, eosinophil; DC, dendritic cell; IL, interleukin; MBP, major basic protein; mono/mϕ, monocyte/macrophage; neu, neutrophil;T,T lymphocyte;TNF, tumor necrosis factor.
Table 1. Chemokine/chemokine receptor nomenclature.The nomenclature was recommended by the IUIS/WHO subcommittee on chemokine nomenclature.
Systematic name Human ligand Chemokine receptor(s) CXC Chemokine/Receptor Family
CXCL1 GROα/MGSA-α CXCR2> CXCR1
CXCL2 GROβ/MGSA-β CXCR2
CXCL3 GROγ/MGSA-γ CXCR2
CXCL4 PF4 Unknown
CXCL5 ENA-78 CXCR2
CXCL6 GCP-2 CXCR1, CXCR2
CXCL7 NAP-2 CXCR2
CXCL8 IL-8 CXCR1, CXCR2
CXCL9 Mig CXCR3
CXCL10 IP-10 CXCR3
CXCL11 I-TAC CXCR3
CXCL12 SDF-1 α/β CXCR4
CXCL13 BCA-1 CXCR5
CXCL14 BRAK/bolekine Unknown
(CXCL15) Unknown Unknown
CXCL 16 CXCR6
C Chemokine/Receptor Family
XCL1 Lymphotactin/SCM-1α/ATAC XCR1
XCL2 SCM-1β XCR1
CX3C Chemokine/Receptor Family
CX3CL1 Fractalkine CX3CR1
CC Chemokine/Receptor Family
CCL1 I-309 CCR8
CCL2 MCP-1/MCAF/TDCF CCR2
CCL3 MlP-1α/LD78α CCR1, CCR5
CCL3L1 LD78β CCR1, CCR5
CCL4 MIP-1β CCR5
CCL5 RANTES CCR1, CCR3, CCR5
(CCL6) Unknown Unknown
CCL7 MCP-3 CCR1, CCR2, CCR3
CCL8 MCP-2 CCR3, CCR5
(CCL9/10) Unknown CCR1
CCL11 Eotaxin CCR3
(CCL12) Unknown CCR2
CCL13 MCP-4 CCR2, CCR3
CCL14 HCC-1 CCR1, CCR5
Remodelling is thought to be the consequence of an aberration of the dynamic process of wound repair that includes matrix production and degradation leading to reconstruction of the tissue. In asthma airway remodelling is described as increased thickening of the airway wall and is thought to be caused by the chronic inflammatory response (5).This reconstruction may lead to irreversible airflow limitation.The various structural alterations seen in asthma include: abnormal epithelium (6;7), thickening of the subbasement membrane (8), alterations in extracellular matrix (9;10), increased vascularisation (11), and increased ASM mass (12;13).
ASTHMA AND ASM
Already in 1868 it was proposed that ASM has a key role in asthma (14). Since the finding of Th2 cells in the airways from asthmatics, it has become widely accepted that asthma is an inflammatory disease, which is reflected in the definition of asthma as described in the guidelines from the Global Initiative for Asthma (15).The idea that inflammation is the cause of asthma led to the idea that ASM cells are passive players in asthma, only at the receiving end of stimuli resulting in enhanced contraction of the airways. As a result of many studies, the paradigm is shifting towards the idea that ASM cells are active players in the inflammatory process and remodelling observed in asthma.Chapter 2 describes classic, current and novel views on the role of ASM cells in asthma and summarizes studies that have led to the changing paradigm. Furthermore, the use of ASM features in phenotyping of patients and the use of (specific) ASM targets in the therapy of asthma are discussed.
ASM and airway inflammation
Airway inflammation in asthma is characterized by infiltration of the airway tissue by eosinophils, T lymphocytes and mast cells (16). Eosinophilic infiltration was long seen as the most characteristic feature of atopic asthma, and many studies have investigated the factors involved in recruitment of eosinophils. Inflammatory cells are recruited through the action of chemotactic cytokines also called chemokines. Previously, chemokines were named after their function or after the cell type by which they were produced.To clarify this complex nomenclature, a systemic system paralleling that of the chemokine receptors (of 7-transmembrane G protein- coupled receptors) has been devised (17). The chemokines have been divided into four families:
the CXC-chemokine family, C chemokine family, CX3C chemokine family, and CC chemokine family.Table 1 summarizes the chemokines known to date categorized by family.
Eosinophils are recruited through the binding of cer tain chemokines including:
CCL11/eotaxin, CCL24/eotaxin-2, CCL26/eotaxin-3, CCL5/Regulated upon Activation, Normal T cell Expressed, and Secreted (RANTES), CCL7/monocyte chemoattractant protein (MCP)-3 and CCL13/MCP-4, and CCL15/leukotactin-1 to CCR3, the CC-chemokine receptor 3. Especially CCL11/eotaxin, CCL24/eotaxin-2 and CCL26/eotaxin-3 are thought to be specific for the recruitment of eosinophils. These chemokines can be secreted by many cell types including: epithelial cells, airway smooth muscle cells, fibroblasts, macrophage cell lines, and mast cells (18-20). CCL11/eotaxin expression was shown to be involved in the early phase of allergen-induced recruitment of eosinophils (21), whereas CCL24/eotaxin-2 and CCL26/eotaxin-3 may be more involved in the late-phase (22;23).
Chapter 1
ROLE OF EPITHELIAL DIFFERENTIATION
IN AIRWAY INFLAMMATION In addition to ASM cells, airway epithelial cells are also resident cells that are affected in asthma. Epithelial damage in asthma is characterized by loss of columnar cells (goblet cells and ciliated cells) (6;34). The loss of columnar cells is repaired by proliferation, migration of basal cells and differentiation of these cells (35). Due to stimuli such as IL-13 that are present in asthmatic airways the basal cells will differentiate into goblet cells (36); this will lead to an epithelium characterized by an increased amount of goblet cells and increased mucus secretion. Airway epithelium is a first line of defence against harmful pathogens by:(i) forming a physical barrier; (ii) producing antimicrobial peptides; and (iii) releasing cytokines and chemokines (37). By releasing cytokines and chemokines the immune system is alerted and immune cells are recruited to the site of infection. Therefore, in chapter 5 we have studied the effect of the Th2 cytokines IL-4 and IL-13 on epithelial differentiation, and how this differentiation affects chemokine synthesis by epithelial cells.
CURRENT THERAPY OF ASTHMA
The established therapy for asthma consists of two classes of drugs: bronchodilators and anti-inflammatory drugs (15). Short- and long-acting β2-adrenoceptor agonists are the most frequently used bronchodilators in the treatment of asthma: the former on an as needed basis, the latter mainly in combination with anti-inflammatory glucocorticoids, which are used for the long-term treatment of asthma. Studies have shown that perhaps β2-agonists also have an anti-inflammatory action, and that combining glucocorticoids with these drugs results in a greater reduction of inflammatory cells. In chapter 6 we have studied whether the combination of the glucocorticoid budesonide and the β2-adrenoceptor agonist for- moterol reduces chemokine release by ASM cells to a greater extent than either drug alone. Although these drugs reduce the symptoms of asthma, they do not cure the disease.
Other strategies to reduce asthma which are not only focused on reducing inflammation are discussed in chapter 2.
SCOPE OF THE THESIS
In summary, this thesis addresses the role of ASM cells in airway inflammation.Chapter 2 first provides an overview of the classic, current and novel view on asthma pathogenesis and gives our perspective on the role of ASM cells in asthma pathogenesis.
Chapter 3 describes the effects of Th2 cytokines and TGF-β, inflammatory mediators that are enhanced in the airways of asthmatic individuals, on release of eosinophil-attracting chemokines by ASM cells. Since CCL11/eotaxin, CCL24/eotaxin-2 and CCL26/eotaxin-3 are involved in different phases of the allergic reaction, we hypothesized that these chemokines would be differentially regulated by Th2 cytokines and TGF-β.
Chapter 4 describes the regulation of the chemokine CXCL8 (IL-8) in ASM cells by the antimicrobial peptides LL-37 and HNP1-3. In addition to its antimicrobial activity, LL-37 has been shown to have immune regulating functions on other cell types including: induction of chemokine release by epithelial cells and induction of chemotaxis of inflammatory cells.
13 12
CCL15 HCC-2/Lkn-1/MlP-1δ CCR1, CCR3
CCL16 HCC-4/LEC/LCC-1 CCR1,CCR2
CCL17 TARC CCR4
CCL18 DC-CK1/PARC/AMAC-1 Unknown
CCL19 MIP-3β/ELC/exodus-3 CCR7
CCL20 MIP-3α/LARC/exodus-1 CCR6
CCL21 6Ckine/SLC/exodus-2 CCR7
CCL22 MDC/STCP-1 CCR4
CCL23 MPIF-1/CKβ 8/CKβ 8-1 CCR1
CCL24 Eotaxin-2/MPIF-2 CCR3
CCL25 TECK CCR9
CCL26 Eotaxin-3 CCR3
CCL27 CTACK/ILC CCR10
CCL28 MEC
GRO, growth related oncogene; PF, platelet factor; ENA, epithelial neutrophils-activating protein; GCP, granulocyte chemotactic protein; NAP, neutrophil-activating peptide; Mig, monokine induced by gamma interferon; IP, interferon-gamma inducible protein; I-TAC, interferon-inducible T cell alpha chemoattractant; SDF, stromal cell-derived factor; BCA, B-cell attracting chemokine; BRAK, breast and kidney expressed chemokine; SCM, single cystein motif; MCP, monocyte chemoattractant protein; MIP, macrophage inflammatory protein; RANTES, Regulated upon Activation, Normal T cell Expressed, and Secreted; HCC, hemofiltrate CC chemokine;TARC, thymus and activation-regulated chemokine; DC-CK, dendritic cell-derived CC chemokine; MDC, macrophage-derived chemokine; MPIF, myeloid progenitor inhibitory factor; TECK, thymus- expressed chemokine; CTACK, cutaneous T-cell attracting chemokine; MEC, mucosae-associated epithelial chemokine.
In this thesis the role of ASM cells in airway inflammation, in particular in the generation of chemokines involved in the recruitment of inflammatory cells into the airway wall, was studied.Chapters 3, 4 and 5 comprise studies in which we have investigated the release of eosinophil-attracting chemokines by ASM cells and airway epithelial cells. In these chapters inflammatory mediators that are thought to play an important role in asthma and other lung diseases such as Chronic Obstructive Lung Disease (COPD) are studied for their effects on chemokine release by ASM cells and epithelium.
INFLAMMATORY MEDIATORS AND ASTHMA
In asthma eosinophilia has been associated with enhanced levels of the Th2 cytokines IL-4 and IL-13, and the pro-fibrotic cytokine TGF-β (24-26).The effect of these mediators on synthetic capacity of ASM cells was studied in chapter 3.
In chapter 4 we examined the effects of antimicrobial peptides (AMPs) on chemokine release by ASM cells. AMPs including the human cathelicidin LL-37 and the human neutrophil defensins (human neutrophil peptides1-3; HNP1-3) are thought to play a role in innate immunity against bacteria but are also known for their immunomodulatory capacity on dendritic cells (DCs) (27;28), inflammatory cells (29;30) and epithelial cells (31-33).These AMPs are expressed by neutrophils and mast cells, cells that are found in or in the vicinity of the ASM layer.
Chapter 1
References
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(8) Roche WR, Beasley R, Williams JH, Holgate ST. Subepithelial fibrosis in the bronchi of asthmatics. Lancet 1989;
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(10) Carroll NG, Perry S, Karkhanis A, Harji S, Butt J, James AL et al.The airway longitudinal elastic fiber network and mucosal folding in patients with asthma. Am J Respir Crit Care Med 2000; 161(1):244-248.
(11) Bousquet J, Jeffery PK, Busse WW, Johnson M, Vignola AM. Asthma. From bronchoconstriction to airways inflammation and remodeling. Am J Respir Crit Care Med 2000; 161(5):1720-1745.
(12) Dunnill MS, Massarella GR, Anderson JA. A comparison of the quantitative anatomy of the bronchi in normal subjects, in status asthmaticus, in chronic bronchitis, and in emphysema. Thorax 1969; 24(2):176-179.
(13) Carroll N, Elliot J, Morton A, James A. The structure of large and small airways in nonfatal and fatal asthma.
Am Rev Respir Dis 1993; 147(2):405-410.
(14) Salter HH. On asthma: its pathology and treatment. 2nd ed. London: Churchill, 1868.
(15) GINA. Global strategy for Asthma Management and Prevention 2006. www.ginasthma.com. 2006.
(16) Ollerenshaw SL,Woolcock AJ. Characteristics of the inflammation in biopsies from large airways of subjects with asthma and subjects with chronic airflow limitation. Am Rev Respir Dis 1992; 145(4 Pt 1):922-927.
(17) Zlotnik A,Yoshie O. Chemokines: a new classification system and their role in immunity. Immunity 2000;
12(2):121-127.
(18) Rothenberg ME. Eotaxin. An essential mediator of eosinophil trafficking into mucosal tissues.
Am J Respir Cell Mol Biol 1999; 21(3):291-295.
(19) Teran LM, Mochizuki M, Bartels J, Valencia EL, Nakajima T, Hirai K et al. Th1- and Th2-type cytokines regulate the expression and production of eotaxin and RANTES by human lung fibroblasts. Am J Respir Cell Mol Biol 1999; 20(4):777-786.
(20) Papadopoulos NG, Papi A, Meyer J, Stanciu LA, Salvi S, Holgate ST et al. Rhinovirus infection up-regulates eotaxin and eotaxin-2 expression in bronchial epithelial cells. Clin Exp Allergy 2001; 31(7):1060-1066.
These properties of LL-37 together with the fact that the ASM layer is infiltrated and surrounded by mast cells and neutrophils in obstructive lung diseases led to the hypothesis that LL-37 and HNP1-3 activate airway smooth muscle function with respect to chemokine production.
By releasing chemokines in response to pathogens, the airway epithelium activates the immune system to defend the lungs against these pathogens. In asthma the airway epithelium is damaged and due to inflammatory mediators present in the airways the differentiation of new epithelial cells may be altered. Previously, chemokine release was mainly studied in undifferentiated cultures of airway epithelial cells.Chapter 5 describes an air-liquid interface culture model in which the effect of epithelial differentiation on chemokine release was studied. We have investigated how epithelial differentiation is influenced by Th2 cytokines and how this affects chemokine release by epithelial cells.
In chapter 6 the effect of a combination of drugs used to treat asthma on chemokine release by ASM cells was studied. As the combination of corticosteroids and β2-agonists has resul- ted in a reduction in asthma symptoms and inflammation in vivo in patients with asthma, we hypothesized that the combination would synergistically inhibit release of CCL11 and CCL26 compared to using corticosteroids alone.
Chapter 1
17 16
(21) Brown JR, Kleimberg J, Marini M, Sun G, Bellini A, Mattoli S. Kinetics of eotaxin expression and its relationship to eosinophil accumulation and activation in bronchial biopsies and bronchoalveolar lavage (BAL) of asthmatic patients after allergen inhalation. Clin Exp Immunol 1998; 114(2):137-146.
(22) Ying S, Robinson DS, Meng Q, Barata LT, McEuen AR, Buckley MG et al. C-C chemokines in allergen-induced late-phase cutaneous responses in atopic subjects: association of eotaxin with early 6-hour eosinophils, and of eotaxin-2 and monocyte chemoattractant protein-4 with the later 24-hour tissue eosinophilia, and relationship to basophils and other C- C chemokines (monocyte chemoattractant protein-3 and RANTES). J Immunol 1999;
163(7):3976-3984.
(23) Berkman N, Ohnona S, Chung FK, Breuer R. Eotaxin-3 but not eotaxin gene expression is upregulated in asthmatics 24 hours after allergen challenge. Am J Respir Cell Mol Biol 2001; 24(6):682-687.
(24) Wills-Karp M, Luyimbazi J, Xu X, Schofield B, Neben TY, Karp CL et al. Interleukin-13: central mediator of allergic asthma. Science 1998; 282(5397):2258-2261.
(25) Wenzel SE, Trudeau JB, Barnes S, Zhou X, Cundall M, Westcott JY et al. TGF-beta and IL-13 synergistically increase eotaxin-1 production in human airway fibroblasts. J Immunol 2002; 169(8):4613-4619.
(26) Redington AE, Madden J, Frew AJ, Djukanovic R, Roche WR, Holgate ST et al.Transforming growth factor-beta 1 in asthma. Measurement in bronchoalveolar lavage fluid. Am J Respir Crit Care Med 1997; 156(2 Pt 1):642-647.
(27) Davidson DJ, Currie AJ, Reid GS, Bowdish DM, MacDonald KL, Ma RC et al. The cationic antimicrobial peptide LL-37 modulates dendritic cell differentiation and dendritic cell-induced T cell polarization. J Immunol 2004;
172(2):1146-1156.
(28) Kandler K, Shaykhiev R, Kleemann P, Klescz F, Lohoff M, Vogelmeier C et al. The anti-microbial peptide LL-37 inhibits the activation of dendritic cells by TLR ligands. Int Immunol 2006; 18(12):1729-1736.
(29) De Y, Chen Q, Schmidt AP, Anderson GM, Wang JM, Wooters J et al. LL-37, the neutrophil granule- and epithelial cell-derived cathelicidin, utilizes formyl peptide receptor-like 1 (FPRL1) as a receptor to chemoattract human peripheral blood neutrophils, monocytes, and T cells. J Exp Med 2000; 192(7):1069-1074.
(30) Tjabringa GS, Ninaber DK, Drijfhout JW, Rabe KF, Hiemstra PS. Human cathelicidin LL-37 is a chemoattractant for eosinophils and neutrophils that acts via formyl-peptide receptors. Int Arch Allergy Immunol 2006;
140(2):103-112.
(31) Van Wetering S, Mannesse-Lazeroms SP, Dijkman JH, Hiemstra PS. Effect of neutrophil serine proteinases and defensins on lung epithelial cells: modulation of cytotoxicity and IL-8 production. J Leukoc Biol 1997; 62(2):217-226.
(32) Van Wetering S,Tjabringa GS, Hiemstra PS. Interactions between neutrophil-derived antimicrobial peptides and airway epithelial cells. J Leukoc Biol 2005; 77(4):444-450.
(33) Aarbiou J, Ertmann M, Van Wetering S, van Noort P, Rook D, Rabe KF et al. Human neutrophil defensins induce lung epithelial cell proliferation in vitro. J Leukoc Biol 2002; 72(1):167-174.
(34) Montefort S, Roche WR, Holgate ST. Bronchial epithelial shedding in asthmatics and non-asthmatics. Respir Med 1993; 87 Suppl B:9-11.
(35) Erjefalt JS, Persson CG. Airway epithelial repair: breathtakingly quick and multipotentially pathogenic. Thorax 1997; 52(11):1010-1012.
(36) Laoukili J, Perret E, Willems T, Minty A, Parthoens E, Houcine O et al. IL-13 alters mucociliary differentiation and ciliary beating of human respiratory epithelial cells. J Clin Invest 2001; 108(12):1817-1824.
(37) Polito AJ, Proud D. Epithelia cells as regulators of airway inflammation. J Allergy Clin Immunol 1998; 102(5):714-718.
Chapter 1
CHAPTER 2
Treating asthma means treating airway smooth muscle (ASM) cells
Suzanne Zuyderduyn1, Maria B. Sukkar2, Anita Fust3, Surinder Dhaliwal3and Janette K. Burgess4,5
1 Department of Pulmonology, Leiden University Medical Centrer, Leiden,The Netherlands 2 Airway Disease Section, National Heart and Lung Institute, Imperial Collega London,
London, United Kingdom
3 Department of Physiology, University of Manitoba,Winnipeg, Canada
4 Respiratory Research Group,Discipline of Pharmacology,University of Sydney,Sydney,Australia 5 Woolcock Institute of Medical Research, Sydney,Australia
Submitted for publication
Abstract
Asthma is characterized by airway hyperresponsiveness (AHR), airway inflammation and airway remodelling. Currently it is thought that airway inflammation is the main cause of AHR and remodelling, and therefore asthma. However, several studies have shown that anti-inflammatory therapy does not reduce AHR, leading to a novel view on the pathogenesis of asthma in which inflammation is no longer thought to be the only cause. In this perspective, we discuss studies that have led to a new view on the role of airway smooth muscle (ASM) in the pathogenesis of asthma in which AHR, remodelling and inflammation are (at least in part) attributable to ASM. Furthermore, we describe how this novel view can influence phenotyping and therapy of patients with asthma.
21
Chapter 2
During the 1990s it was found that eosinophilic inflammation was a characteristic of airways in many asthmatic patients (2) which led to the current view that eosinophils are the main cause of asthma. A great deal of effort went into developing strategies to reduce eosinophilic inflammation in the airways. Migration and survival of eosinophils is dependent on many mediators including immunoglobulin E (IgE) and T helper 2 (Th2) cytokines (IL-4, IL-5 and IL-13), and many studies have investigated targeting these molecules. Studies with an anti-IgE antibody (omalizumab) have shown reduction in allergen-induced eosinophil numbers in sputum and peripheral blood (3), and reductions in eosinophils and T cells in bronchial biopsies of asthmatics (4). Despite these anti-inflammatory actions and a reduction in exacerbations (5;6), no improvement in AHR was seen after treatment (4;7). Another trial investigating the effects of an anti-IL-5 monoclonal antibody in asthmatics also showed no reduction in AHR despite reduced inflammatory cell numbers (8). These data imply that reductions in both eosinophilia and exacerbation frequency are not accompanied by improvements in AHR, and therefore that the links between (i) inflammation and AHR; and (ii) exacerbation rate and AHR are not as clear as was previously thought. In addition, studies correlating inflammation with AHR have contradictory results. Correlations between inflammatory cell numbers and the degree of AHR have been published, however an equal number of studies have shown an absence of any correlation (9-11), suggesting that AHR cannot be explained by inflammation alone.
The apparent dissociation between inflammation and AHR has led to the novel view suggesting that inflammation and AHR are separate entities in asthma (Figure 1, novel view).
Both inflammation and AHR can induce symptoms in patients, and treating only one of them will not reduce all of the symptoms of asthmatic patients. However, inflammation may be partly responsible for AHR since mediators released by inflammatory cells are known to induce contraction of airway smooth muscle cells. Furthermore, the immunology of asthma is complex with many different cytokines, chemokines and lipid mediators involved in the different aspects of the disease (12), but their roles are not distinct as many cytokines and chemokines have overlapping biological activities (redundancy). Eosinophil recruitment, for instance, is mediated by a variety of cell-bound and soluble mediators including adhesion molecules, cytokines (e.g. IL-5), and chemokines (CCL11 [eotaxin], CCL24 [eotaxin-2], CCL26 [eotaxin-3], and CCL5 [Regulated upon Activation, Normal T cell-Expressed and Secreted, RANTES]). Knocking out only one of these molecules will not lead to a complete reduction in eosinophilic inflammation.Therefore, anti-cytokine strategies targeting multiple mediators may prove to be more successful, and this type of strategy should not be dis- carded. Moreover, the lack of effect on AHR of some anti-cytokine treatments does not exclude effects of these treatments on other parts of the disease such as remodelling.
Unfortunately, studies designed to examine the long term effects of anti-cytokine treatment on the development of remodelling have not yet been performed.
THE ROLE OF ASM CELLS IN ASTHMA:
CLASSIC, CURRENT AND NOVEL VIEW Airways from asthmatic subjects are more responsive to various stimuli that do not provoke a large constrictive effect in healthy subjects. Airway smooth muscle (ASM) cells mediate contraction of the airways, and in the 1950s and 1960s it was thought that an exaggerated contractile response of these cells was the cause of asthma (Figure 1, classic view). Asthma is currently defined as a chronic inflammatory disorder characterized by reversible airways obstruction and airway hyperresponsiveness (AHR). In the current view, inflammation causes the symptoms of asthma directly and indirectly by inducing contraction of ASM and by inducing changes in structural components of the airway wall (including ASM cells) leading to airway remodelling, thereby implying that inflammation is the main cause of asthma (Figure 1, current view). A novel view on the pathogenesis of asthma is arising from studies showing that ASM cells are not only involved in contraction, but also in the remodelling and inflammation of the airways that is observed in asthma. In this view we propose that AHR, remodelling and inflammation are all attributed (at least to some extent) to ASM (Figure 1, novel view).
In the following perspective, we will discuss studies that have led to this new view, and how this view may alter phenotyping of asthma and direct development of novel intervention strategies.
INFLAMMATION IS NOT THE ONLY CAUSE
OF AIRWAY HYPERRESPONSIVENESS IN ASTHMA Asthma is a chronic inflammatory disorder characterised by variable excessive airway narrowing and airway hyperresponsiveness (AHR). ASM cells are thought to be the major effector cells of airway narrowing although other factors, such as swelling of airway wall compartments and mucus plugging, may amplify the narrowing (1).The current view is that inflammation is the main underlying mechanism of airway narrowing and perhaps also AHR.
Chapter 2
Figure 1. Schematic representation of the classic, current and novel view on the role of ASM in asthma. In the classic view ASM is triggered to produce symptoms. Upon the finding of eosinophilic inflammation in many asthmatic patients the current view was established in which inflammation leads to AHR, remodelling and subsequently symptoms. In the novel view, ASM cells are involved in AHR, remodelling and inflammation, and these three features will lead to symptoms of asthma.
ASM, airway smooth muscle; AHR, airway hyperresponsiveness
CLASSIC VIEW CURRENT VIEW NOVEL VIEW
dependence may be caused by inflammation which could lead to uncoupling of the airways and parenchyma, or by oedema which leads to thickening of the airways and thereby decreases radial forces acting on the airways during a deep inspiration (22;23). Anti-inflammatory therapy (a high dose of prednisone) improved bronchodilation after a deep inspiration in asthmatics, suggesting that inflammation indeed impairs airway mechanics (24), and is partly responsible for AHR. A recent study suggests that the impaired bronchodilatory effect of DI in intermit- tent and mild persistent asthmatics is a result of inflammatory mechanisms within the ASM bundle and airway wall, as increased numbers of mast cells within the ASM bundles and CD4+T cells in the lamina propria were associated with the reduced bronchodilatory effect (25).The studies discussed suggest that inflammation is not the only cause of AHR, but may be partly responsible.
AIRWAY SMOOTH MUSCLE CELLS ARE
A PART OF THE INFLAMMATORY PROCESS IN ASTHMA In the classic and current view ASM cells are considered to be passive players that only respond to stimuli by contracting; the secretory capacity of ASM cells was not recognized until recently. Studies using cultures of ASM cells isolated from lung tissue (trachea, bronchi) either by enzymatic digestion or the explant method led to the novel view that ASM cells are active players in inflammation.
ASM phenotype
Freshly isolated ASM cells are contractile, but upon culture in serum-rich conditions ASM cells modulate from a “contractile” phenotype to a “synthetic-proliferative” phenotype which lacks responsiveness to contractile agonists and has reduced expression of contractile proteins such as smooth muscle myosin heavy chain (smMHC), smMLCK, and smooth muscle α-actin (αSMA).These cells are however highly proliferative in response to mitogens and produce extracellular matrix (ECM) proteins and cytokines (26). “Synthetic-proliferative” cells can mature into “contractile” cells during lung development in dogs and this process can be mimicked in vitro by prolonged serum deprivation of canine ASM cells leading to a “hyper- contractile” phenotype with increased expression of αSMA, smMHC, SM22, desmin, calponin and M3 muscarinic receptor (27-30). This modulation and maturation is known as phenotype switching (see Figure 2). Whether this switching from “contractile” to “synthetic- proliferative” also takes place in vivo in humans remains to be established. Furthermore, the existence of these two phenotypes of cells in vivo and whether the ratio of “contractile”
versus “synthetic-proliferative” cells contribute to functional abnormalities needs further investigation. The contractile function of ASM cells was long seen as the most important function of these cells. However, studies with “synthetic-proliferative” cells have shown that ASM cells are a source of a wide variety of inflammatory mediators, including cytokines (e.g. IL-6), lipid mediators (e.g. prostaglandin E2), ECM (e.g. collagen, fibronectin) and chemokines (e.g. CCL11, CXCL8 [IL-8], CXCL10 [IP10], CX3CL1 [fractalkine]) (reviewed in (31;32)). By secreting chemokines, ASM cells can contribute to the formation of a chemokine gradient leading to migration of inflammatory cells from the blood vessels into
25 24
ASM: the bad guy in asthma?
If inflammation is not the main cause of AHR, what is? AHR is defined as exaggerated airway narrowing to non-specific irritants or pharmacological agonists, which is reversible by bronchodilators that relax ASM.This definition implies that ASM is the “bad guy”, and many studies have focussed on the nature of the change in ASM from asthmatics. Previously, it was thought that the ASM from asthmatics could generate more force, and therefore would contract to a greater extent. However, no direct evidence for this has been found.
Stephens et al. showed that the force generation of allergen-sensitized ASM was equal to control muscle (13), suggesting that there is no difference between allergic and non-allergic smooth muscle. Due to the lack of evidence of intrinsic abnormalities in ASM, alternative explanations have been put forward. The first hypothesis is that increased ASM mass can generate more force and therefore more narrowing (14). The second hypothesis is that reduced load on ASM leads to enhanced narrowing. Decreased coupling between ASM and surrounding tissue (“uncoupling”) which is the result of inflammation (oedema) and remodelling (loss of alveolar attachments) could cause the reduction in load (15;16).The third hypothesis suggests that not force, but shortening velocity and capacity are altered in asth- matics implying that ASM from asthmatics has an intrinsic difference after all. Studies with smooth muscle from antigen-sensitized dogs have shown that this smooth muscle has a greater maximum shortening velocity (V0) and capacity (Lmax) (17). A study using human bronchial smooth muscle cells from asthmatics reported that these cells have increased maximum shortening capacity and velocity (18).The molecular mechanism underlying increased maxi- mum shortening velocity has been investigated. Sensitization of dog trachealis resulted in increased quantity and activity of myosin light chain kinase (MLCK) (13). Enhanced MLCK activity results in enhanced phosphorylation of myosin light chain (MLC). This allows acto- myosin adenosine triphosphatase (actomyosin ATPase) to be activated by actin, thereby ini- tiating contraction via crossbridge cycling (19;20). As in dog ASM, smooth muscle MLCK (smMLCK) content in human sensitized ASM was increased. Subsequently, it was also found that smMLCK mRNA levels were increased in asthmatic ASM compared to normals (18).
These studies point to a causal role for an intrinsic abnormality in ASM in the pathogene- sis of asthma.
ASM and deep inspirations
Another abnormality seen in asthmatics is that their airways respond differently to stretch by deep inspirations (DI) compared to airways of healthy subjects. In normal subjects DI lead to bronchodilation and bronchoprotection, whereas in asthmatics DI do not reduce bronchoconstriction and may even enhance obstruction. When DI were prohibited in nor- mal subjects prior to methacholine challenge tests, these subjects became hyperresponsive suggesting that DI protect the lungs from bronchoconstriction in normal subjects (21).
Whether this abnormality is due to changes in ASM cells or due to other factors is not known, but Skloot and Togias suggested that DI lead to ASM stretch which results in relaxation (21). Due to stiffer airways, reduced force of ASM or loss of interdependence of parenchyma and airways, asthmatics may be less responsive to stretching of the ASM. Loss of inter-
Chapter 2
ASM secretory function
A role for ASM cells as secretory cells involved in recruitment of inflammatory cells is further highlighted by studies showing intra-ASM inflammation. Ammit et al. have shown that more mast cells were present in the smooth muscle of human sensitized bronchi compared to non-sensitized bronchi (35). Brightling et al. reported that submucosal eosinophil and mast cell numbers did not differ between patients with asthma and eosinophilic bronchitis (characterized by infiltration of the airways by eosinophils without AHR), nor did the thickness of the BM or the lamina reticularis. However, when the ASM layer was assessed, higher numbers of mast cells infiltrating this layer were seen in asthmatics compared to the patients with eosinophilic bronchitis (36). Furthermore, in asthmatic patients a correlation was found between number of mast cells and PC20 methacholine (provocative concentration of methacholine that results in a 20% reduction in forced expiratory volume in one second [FEV1]; measure of AHR): higher numbers of mast cells were associated with a lower PC20 which suggests that the mast cells may influence AHR in asthma. However, it was not shown whether this was specific for asthma or more general for obstructive lung diseases. The correlation between intra-ASM mast cell numbers and AHR in asthmatics suggests that mast cells in the ASM bundles are responsible for enhanced airway narrowing seen in asthmatics.
Perhaps reducing intra-ASM inflammation is more useful than reducing mucosal inflammation.
Reducing mast cell recruitment by ASM cells or stabilizing mast cells to prevent degranulation are two possible options in this respect.
With the discovery of intra-ASM inflammation, the altered secretory pattern of ASM from asthmatics and the putative interactions between mast cells and ASM cells have become the focus of many studies. It is thought that the ASM is responsible for the recruitment of the mast cells by secretion of mast cell-chemotactic chemokines. Several studies have shown that ASM cells can release CCL11 (37-39), CXCL10 (40;41), stem-cell factor (SCF) (42), CXCL12 (stromal cell-derived factor-1 [SDF-1]) (40) and CX3CL1 (43) in response to various stimuli. These chemokines are all chemotactic for mast cells and their role in mast cell chemotaxis towards asthmatic ASM cell supernatant has been studied (40;44;45). Chan et al. have shown that asthmatic ASM cells produce more CCL11 constitutively and upon stimulation with the Th2 cytokine IL-13 compared to non-asthmatic ASM cells (46). In a study by Brightling et al. stimulation of ASM cells from asthmatics with pro-inflammatory and Th1 cytokines led to release of increased amounts of CXCL10 compared to ASM cells from normals, whereas no difference was found between asthmatics and normals with respect to CCL11, CXCL12 and CXCL8 release (40). Sutcliffe et al. found that upon Th2 cytokine stimulation asthmatic ASM cell supernatants were more chemotactic for mast cells, which could not be explained by enhanced expression of chemokines including CCL11, CXCL10, SCF or TGF-β (44).The authors found an alternative explanation for the enhanced chemo- tactic activity of asthmatic ASM cell supernatants: these supernatants lack (an) inhibitory factor(s) which is expressed by non-asthmatic ASM supernatants. The nature of the factor(s) is however not clear.
the surrounding tissues of the airways.
Recent technological advances, including the optimization of the establishment of ASM cultures from endobronchial biopsies, have led to studies investigating the differences between ASM cells obtained from asthmatics (the so-called asthmatic ASM cells) and ASM cells obtained from non-asthmatics. These studies have shown that asthmatic ASM cells are intrinsically different from non-asthmatic ASM cells: they proliferate faster and have an altered secretory pattern (33;34). Perhaps these cultures represent the phenotype of the cells in vivo.
Chapter 2
Figure 2.Phenotype switching of ASM cells.ASM cells can modulate from a “contractile” to a “syn- thetic-proliferative” phenotype which shows reduced expression of contractile proteins and increased proliferation and synthetic capacity. “Synthetic-proliferative” cells can mature into “contractile cells”
during lung development.
ASM, airway smooth muscle
PHENOTYPE SWITCHING
AIRWAY SMOOTH MUSCLE REMODELLING:
CAUSE OR CONSEQUENCE OF ASTHMA?
In addition to inflammation, remodelling of the airways is also a major histopathological feature of asthma. Remodelling is thought to be the consequence of an aberration of the dynamic process of wound repair that includes matrix production and degradation leading to recon- struction of the tissue. Due to unknown circumstances this process is disturbed in asthma and leads to enhanced production of matrix leading to fibrosis of the tissue. In asthma airway remodelling is described as increased thickening of the airway wall due to various structural alterations (61). These alterations which could have profound physiological consequences are summarized in table 1.
Table 1. Structural alterations contributing to airway remodelling
Alterations Reference
Abnormal epithelium (10;62;63)
Subbasement membrane thickening (64)
Alterations in interstitial matrix (65;66)
Increased vascularisation (67)
Alterations in mucous glands/enhanced mucus production (68)
Increased smooth muscle mass (69)
The classic and current view on remodelling states that remodelling is a consequence of the chronic inflammatory response that develops during the disease. However, remodelling can already be found in young children before the onset of clinical asthma. In a study by Pohunek et al. the thickness of the lamina reticularis (=subbasement membrane) was already increased two years prior to the development of clinical asthma (70). Furthermore, no correlation was found between duration of the disease and the degree of collagen deposition in the lamina reticularis suggesting that airway fibrosis evolves in parallel or even precedes eosinophilic inflammation. In another study in children with asthma, thickening of the lamina reticularis was seen in 9 out of 10 children, whereas eosinophilic inflammation was only seen in 1 out of 10 children (71). In studies in adult patients it was found that the thickness of the lamina reticularis increased with the severity of the disease (72), but was not related to duration of the disease (73).Therefore, airway remodelling may exist prior to development of inflammation and clinical asthma and may be a cause of asthma. In this perspective we will focus on remodelling of the airway smooth muscle layer.
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The interactions between mast cells and ASM cells are also being scrutinized. Cell-cell contact between these cells may be important for a functional interaction (47). Mast cells adhere to ASM cells in part via a molecule known as tumor suppressor in lung cancer-1 (TSLC-1) (48).
Upon adhesion the mast cells may release mediators including histamine, prostaglandin D2 (PGD2), and leukotriene C4 (LTC4) which can induce contraction of ASM. Mast cell tryptase is also an important activator of ASM; it can induce cytokine release (49), induce proliferation of ASM cells (49-51), and potentiate the contractile response to histamine (52).
In summary, the secretory function of ASM cells may be very important in the recruitment of mast cells into the ASM bundle, and could lead to mast cell activation and subsequent alterations in ASM contractility and remodelling.
ASM cell surface molecules and immunomodulatory function
In addition to its secretory function, ASM cells also express many cell surface molecules indicating that they may directly interact with immune cells or may have inherent immune functions of their own. Integrins (intercellular adhesion molecule [ICAM]-1 and vascular cell adhesion molecule [VCAM]-1) and CD44 are important for the adhesion of T cells to ASM cells; adhesion between activated T cells and ASM cells leads to DNA synthesis in the ASM cells (53). We have reported that ASM cells express OX40 ligand (OX40L), a member of the TNF superfamily expressed on various inflammatory cells including B cells and dendritic cells, and ligation of this molecule led to IL-6 release (54). Major histocompatibility complex (MHC) class II and the costimulatory molecules CD40 (55;56), CD80 and CD86 (57) are also present on ASM cells. Despite the expression of MHC class II the ASM cells are not able to present antigen (56), however ASM cells and activated T cells can interact via cell adhesion molecules and costimulatory molecules (57).
Recently, expression of Toll-like receptors (TLRs) was detected on ASM cells and their expression was shown to be increased upon stimulation with pro-inflammatory stimuli (58).
TLRs are pattern-recognition receptors critically involved in activation of innate and adaptive immune responses. Activation of TLR2,TLR3, and TLR4 on the ASM cells by their respective ligands led to CXCL8 and CCL11 release, indicating that these TLRs are functional.
Furthermore, ASM cells respond to the TLR3 ligand dsRNA (a viral replicative intermediate) by releasing CXCL10 which could lead to recruitment of mast cells (59). TLR ligands have also been shown to amplify pro-inflammatory interactions between ASM cells and peripheral blood mononuclear cells by augmenting cytokine and chemokine production by these cells under co-culture conditions (59;60).These data show that ASM cells are able to interact with infiltrating immune cells, and may also potentially be involved in innate and adaptive immune mechanisms that underlie airway inflammatory responses.
Chapter 2
Altered extracellular matrix
In addition to increased ASM mass, alterations in the extracellular matrix (ECM) contribute to the thickening of the airway wall. In asthmatics the ECM is altered compared to that of healthy subjects: increased deposition of collagen I, III,V, fibronectin, tenascin, hyaluronan, versican and laminin is found (81-83), whereas collagen IV and elastin deposition is decreased (84).
The composition of the ECM is tightly regulated by a balance between de novo synthesis and degradation. Matrix metalloproteases (MMPs) are responsible for degradation of ECM components; these proteases are secreted in an inactive form and can be activated by other proteases such as trypsin. They are counter-regulated by their specific inhibitors named tissue inhibitors of metalloproteases (TIMPs). In asthmatics the balance between MMPs and TIMPs may be altered; mucosal biopsies from asthmatic patients have shown enhanced MMP-9 mRNA expression in eosinophils (85), and enhanced MMP-2 and MMP-9 levels have been detected in sputum from asthmatics (86;87). Furthermore, inhaled corticosteroids have been shown to decrease subepithelial collagen deposition by reducing MMP-9 expression and increasing TIMP-1 expression in bronchial biopsies from asthmatics (88). Genomic studies have shown that another type of protease, ADAM (A Disintegrin And Metalloprotease) -33 which is mainly expressed by ASM cells, is linked to asthma and AHR (89). Recently, other members of the ADAM protease family including ADAM-8, -9, and -12, and some of their Increased ASM mass
Increased airway smooth muscle mass was first described by William Stirling in 1878 in lungs from cats that were infected by a nematode worm. He described “inter-alveolar hypertrophy due to a great increase in the number of the non-striped muscular fibres”(see Figure 3) (74).
Huber and Koessler described increased airway smooth muscle mass in patients with asthma in 1922 (75) and in 1969 Dunnill et al. published a paper showing that the amount of ASM in lung tissue was increased in patients who died in status asthmaticus compared to normal individuals who had died suddenly with no previous history of chronic bronchitis, and to patients who had died with a history of chronic obstructive lung disease without emphysema (chronic bronchitis) or with emphysema (68). The degree of ASM mass increase seems to be related to the severity of asthma (76-78), and even in young asthmatics (17-23 years old) more ASM mass is detected when compared to age-matched controls (73).These data suggest that increased ASM mass is not the consequence of asthma. The novel view suggests that increased ASM mass is present before symptoms of asthma develop and therefore it may be (one of) the cause(s) of asthma.
The increase in ASM mass may result from: more muscle mass to begin with, an increase in cell size (hypertrophy) or in cell number (hyperplasia) due to growth factors, decreased apoptosis of ASM cells, recruitment of stem cells or transformation of mesenchymal cells (79). Hyperplasia and hypertrophy of ASM have been the focus of many studies. Ebina et al.
have shown hyperplasia of ASM in subjects with increased ASM mass restricted to central airways, whereas both hyperplasia and hypertrophy were found in patients with increased ASM in central and peripheral airways, suggesting that both hyperplasia and hypertrophy can contribute to increased ASM mass (80). Other investigators have shown either hypertrophy (76) or hyperplasia (78) of ASM in asthmatic airways.
Studying airway smooth muscle mass in young children that have not developed clinical asthma would show us whether increased ASM mass precedes asthma or whether it is a consequence of hyperplasia or hypertrophy caused by the increased presence of growth factors. Due to ethical considerations, performing bronchial biopsies in children to answer this question is difficult.
Chapter 2
Figure 3.Hypertrophy of non-striped smooth muscle fibres. Drawings made by William Stirling.
Figure reproduced from (74)with permission from the publisher.
Figure 1. Figure 2. Figure 3.
addition, the ECM can influence migration of ASM cells. Increased migration of ASM cells was seen when membranes were coated with collagen III,V and fibronectin compared to collagen I, elastin and laminin (101). Whether migration of ASM cells plays a role in vivo needs to be established.
In summary, the existence of airway remodelling before the clinical onset of asthma suggests that remodelling is perhaps (one of) the cause(s) of asthma. Increased ASM mass due to proliferation of ASM cells, altered ECM deposition by ASM cells or migration of ASM cells, is correlated to severity of asthma but not duration of asthma suggesting that the thickening is not the result of asthma but may be the cause.
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inhibitors (TIMP-1 and TIMP-3) have been reported to be overexpressed in sputum cells from asthmatics (90) suggesting that these proteases are involved in the pathogenesis of asthma.
In addition to providing support to tissue, ECM has been shown to modulate cell development, migration, and proliferation (67). The composition of the ECM on which ASM cells grow influences their proliferation rate as was shown by Hirst et al (91). ASM cells grown on fibronectin or collagen I proliferated faster in response to mitogens than cells grown on plastic alone, whereas laminin reduced the proliferation rate of ASM cells. ASM cells from asthmatics have been shown to produce an altered array of ECM proteins compared to ASM cells from non-asthmatics; they produce more perlecan and collagen I and less laminin-alpha1 and collagen IV (92). Enhanced ECM production by asthmatic ASM cells was shown to be the result of increased production of connective tissue growth factor (CTGF) (93).This altered ECM produced by the asthmatic ASM cells enhanced the proliferation rate of ASM cells (asthmatic or non-asthmatic) grown on it, suggesting that ASM cells, by producing ECM proteins, may themselves influence airway remodelling. In addition, asthmatic ASM cells can influence vessel formation in and around the ASM bundle as CTGF produced by ASM cells anchors vascular endothelial growth factor (VEGF) to the ECM (94).
How ECM proteins induce proliferation is not known, however a study from Freyer et al.
has shown that ECM proteins can influence survival of ASM cells. When ASM were grown on plastic and were treated with the protein synthesis inhibitor cyclohexamide (CHX) all cells became apoptotic; growing ASM cells on fibronectin, collagen I or IV or on laminin reduced apoptosis (95).These data show that the ECM profile is very important for the proliferation and survival of ASM cells.
A recent report by Chan et al. has shown that the altered secretion of ECM components from asthmatic ASM cells leads to enhanced eotaxin expression, suggesting that the ECM may also influence the synthetic capacity of ASM cells (46). In addition, ECM proteins are important for phenotype maintenance. Endogenously expressed laminin is required for maturation of ASM cells into a contractile phenotype (96).
These data suggest that ECM produced by ASM cells is important in remodelling of the airways, as the ECM profile determines the proliferation, synthetic capacity and phenotype of ASM cells.
Increased ASM migration
The presence of an increased smooth muscle layer and “smooth muscle-like cells” or
“myofibroblasts” outside the smooth muscle cell compartment has given rise to the novel idea that ASM cells can migrate within the airway wall. There are several views on what
“myofibroblasts” may be. One view is that they originate from fibroblasts and take part in the growth, development and repair of tissues (97). It is also thought that myofibroblasts are fibrocytes recruited from the blood (98), or that they originate from epithelial cells that have undergone transition into mesenchymal cells (epithelial-mesenchymal transition, EMT) (99).
Another theory is that they are recruited smooth muscle cells that have migrated from the bundles (100). ASM cells in vitro have the capacity to migrate in response to a growing list of mediators including growth factors (101), cysteinyl leukotrienes (102) and cytokines (103). In
Chapter 2
Figure 4.Asthmatic ASM cells are hypercontractile, hyperproliferative, and hypersecretory. Asthmatic ASM express more MLCK which may be attributable to the lack of c/EBPα; this may lead to enhanced contractility of cells. Hyperproliferation of asthmatic ASM cells is thought to be the con- sequence of the lack of c/EBPα and the altered ECM in which the cells are embedded.The hyper- secretion by asthmatic ASM cells may be caused by the altered ECM and by the lack of c/EBPα which would result in enhanced transcription of inflammatory genes including CCL11 and CXCL10.
ASM, airway smooth muscle; MLCK, myosin light chain kinase; c/EBP, CCAAT-enhancer binding protein; ECM, extracellular matrix; CCL; CC chemokine ligand; CXCL, CX chemokine ligand.
FEATURES OF ASTHMATIC ASM CELLS
ASTHMATIC ASM CELLS:
IS CCAAT-ENHANCER BINDING PROTEIN (C/EBP)α THE PROBLEM?
Asthmatic ASM cells have been shown to be intrinsically different from ASM cells from non-asthmatics: they are hypercontractile, hyperproliferative, and hypersecretory (Figure 4).
Why the asthmatic ASM cells have different contractile, proliferative and secretory capaci- ties is not known, however recent studies point to a role for the transcription factor c/EBPα. Cultured asthmatic ASM cells were shown to lack the anti-proliferative isoform of the CCAAT-enhancer binding protein (c/EBP)α (104;105).This transcription factor is impor- tant in many processes in ASM cells. Increased MLCK expression is thought to be respon- sible for the increased contractility in asthma (see figure 5).The promoter of the MLCK gene has many c/EBPα binding sites. c/EBPα is a negative regulator, suggesting that lack of c/EBPα observed in asthmatic ASM cells (105;106) may result in enhanced expression of MLCK and may therefore be important in AHR.
The abnormality resulting in increased ASM mass is thought to be the lack of the anti- proliferative c/EBPα (104). C/EBPα is an important regulator of proliferation through the regulation of the cell cycle inhibitor p21waf/cip1. Asthmatic ASM cells lack the anti-prolifera- tive isoform of c/EBPα which results in enhanced proliferation. In addition, steroids also exert their effect via c/EBPα through an interaction of the glucocorticoid receptor (GR) and c/EBPα that activates p21 (107). Due to the lack of c/EBPα these drugs are not very potent in inhibiting proliferation of ASM cells.
A recent review by Borger et al. describes the role of c/EBPα in airway inflammation (106).
In short, c/EBPα can silence the inflammatory response through interference with nuclear factor kappa B (NFκB)-driven gene expression; a lack of c/EBPα will result in more expres- sion of NFκB-dependent inflammatory genes.
The lack of c/EBPα seems a likely candidate for the explanation of the altered contractility, proliferation and synthetic capacity in asthmatic ASM cells.Whether the lack of c/EBPα or other intrinsic differences between ASM cells from asthmatic individuals and non-asthmatic individuals are the cause of asthma remains to be established. Studies focussing on identifying and targeting the differences will lead to a better understanding of the role of the ASM cell in asthma.
Chapter 2
Figure 5.Schematic representation of intracellular pathways involved in contraction of ASM cells.
A balance in favour of MLCK results in enhanced phosphorylation of myosin leading to enhanced contraction.The balance between MLCK and MLCP could be a target for intervention. Reducing MLCK or enhancing MLCP will result in less contraction.
ASM, airway smooth muscle; MLCK, myosin light chain kinase; MLCP, myosin light chain phosphatise;
GPCR, G protein coupled receptor; PLC, phospholipase C; IP3, inositol triphosphate; DAG, diacyl glycerol; Ca2+, calcium; PKC, protein kinase C; ROCK, Rho kinase.
Phenotyping based on ASM characteristics
Considering the novel view on the role of ASM cells in AHR, remodelling and inflammation, these cells should be studied more thoroughly and their phenotype and function taken into account when patients are classified into a specific phenotype. In particular, the amount of ASM mass, mast cell infiltration of the ASM layer and the “contractile” versus “synthetic-proliferative”
phenotype of ASM cells should be considered when classifying patients, as these features may correlate to severity and AHR. The degree of ASM bulk and mast cell infiltration could be measured in bronchial biopsies of asthmatics; the phenotype of ASM could perhaps be studied by assessing staining for specific contractile proteins and relating them to severity and AHR.
Increased ASM mass is now being extensively studied by several research groups that are trying to answer the question whether patients with different degrees of severity of disease can be distinguished by specific airway structural components. When patients with intermittent, mild to moderate and severe asthma were compared to healthy controls and patients with COPD, size of ASM cells and fibroblast accumulation under the BM were higher in severe asthmatics compared to the other patient groups (78). Furthermore, MLCK content in ASM from severe asthmatics was enhanced and was negatively correlated with pre- and post- bronchodilator FEV1. This study shows that quantification of components of the airway architecture allows the discrimination between severe and milder disease. In a second study comparing severe to moderate asthmatics, an increase in ASM area seemed to be the best marker for severity, as ASM area was negatively correlated to FEV1in severe asthmatics but not in the moderate asthmatics (77). In addition, these investigators found a reduced distance between ASM area and the epithelium, and more IL-8 and eotaxin in the ASM area in bronchial biopsies from severe asthmatics compared to subjects with moderate asthma. In a third study on ASM area by Woodruff et al., mild to moderate asthmatics were compared to healthy controls with respect to ASM content, cell volume, cell number and gene expres- sion for contractile proteins (76). Increased ASM volume was found in the asthmatic patients, which was reflected by an increase in cell number rather than cell size. In this third study no differences in gene expression for contractile proteins were found, suggesting that AHR in asthma is mainly dependent on the amount of ASM, and not on increased expression of contractile proteins.Together, these three studies suggest that ASM mass due to hyperplasia or hypertrophy reflects severity of disease, and therefore the amount of ASM mass could be used as a marker of severity of disease. However, the studies performed so far have not prospectively addressed whether ASM mass relates to severity of disease.
This concept of clinical assessment is however challenged as it is not known how specific increased ASM mass is for asthma. Hogg et al. have shown that ASM area is also enhanced in small airways of COPD patients with GOLD (Global initiative for chronic Obstructive Lung Disease) status 3 and 4 (114). In this study thickening of the airway walls (in part explained by increases in ASM mass) had the strongest association with progression of COPD, suggesting that remodelling and increases in ASM mass are not specific for asthma but are a feature of progressive lung disease. The role of ASM cells in COPD is less extensively studied. In the large airways alterations in ASM mass were not observed (115),
37 36
ASM CHARACTERISTICS SHOULD BE USED
TO PHENOTYPE PATIENTS WITH ASTHMA As ASM cells from asthmatic individuals are different to normals and these cells are involved in AHR, remodelling and inflammation, we propose that studying these cells in patients will lead to better phenotyping and treatment of patients.
Phenotyping of asthma
Asthma is a complex disease, and patients with asthma can have different symptoms and respond differently to treatment. Therefore, phenotyping of patients might result in the development of more specific therapies leading to better long term outcomes. Currently, patients are separated into different classes according to: severity of disease, aetiology, clinical pattern, immunological findings, and pathophysiology.The most commonly used classification is the one according to severity of disease.The severity of disease is based on the symptoms of the patient, the amount of β2-agonist reliever medication used by the patient and lung function parameters (108), leading to categorization into intermittent and three levels of persistent disease (mild, moderate and severe). Wardlaw et al. suggest that objectively measurable terms should be used to phenotype patients (109) rather than symptoms which rely on subjective measures. For instance the pathophysiology could be used since both the pathology (amount of inflammation, remodelling) and physiology (lung function, AHR, reversible versus fixed airflow obstruction) can be measured.
Including immunological findings may also help to develop better treatments for asthma.
During the 1990s it was found that eosinophilic inflammation was a characteristic of many asthmatic airways (2), which led to the idea that eosinophils were the cause of asthma.
However, this is challenged by the fact that in many patients no eosinophilia is found in spu- tum samples during exacerbations (110;111). Non-eosinophilic asthma is found in 25% of patients with symptomatic asthma and in up to 50% of patients with asthma using high doses of steroids (112;113); it is associated with neutrophilic inflammation, suggesting that the neutrophils may be the bad guys in patients with this type of asthma. If the type of inflam- mation in the airways of the patient is taken into account, more specific treatments for each phenotype can be developed, which may allow better management of individual patients.
The drawback to this strategy is that, in practice, routine measurements of pathology and immunology would have to be added to the more standard measurement of lung physiology.
This may prove to be very difficult since the measurement of pathology and immunology require more invasive techniques (sputum, bronchoalveolar lavage, biopsies) than measuring physiology alone, and performing all these measurements would be very time consuming.
Therefore, finding non-invasive markers of inflammation and remodelling is very important.
Chapter 2
