REVIEW
Arch. Immunol. Ther. Exp., 2009, 57, 331–344 PL ISSN 0004-069X DOI 10.1007/s00005-009-0039-4
Immunopathogenesis of bronchial asthma Milan Buc1, Martin Dzurilla1, Mojmir Vrlik2 and Maria Bucova1 1 2
Department of Immunology, Comenius University School of Medicine, Bratislava, Slovakia Martin Immunology Centre, Martin, Slovakia
Received: 2009.01.15, Accepted: 2009.04.16, Published online: 2009.08.18 © L. Hirszfeld Institute of Immunology and Experimental Therapy, Wroc³aw, Poland 2009
Abstract Bronchial asthma is a common immune-mediated disorder characterized by reversible airway inflammation, mucus production, and variable airflow obstruction with airway hyperresponsiveness. Allergen exposure results in the activation of numerous cells of the immune system, of which dendritic cells (DCs) and Th2 lymphocytes are of paramount importance. Although the epithelium was initially considered to function solely as a physical barrier, it is now evident that it plays a central role in the Th2-cell sensitization process due to its ability to activate DCs. Cytokines are inevitable factors in driving immune responses. To the list of numerous cytokines already known to be involved in the regulation of allergic reactions, new cytokines were added, such as TSLP, IL-25, and IL-33. IgE is also a central player in the allergic response. The activity of IgE is associated with a network of proteins, especially with its high- and low-affinity Fc receptors. Understanding the cellular and molecular mechanisms of allergic reactions helps us not only to understand the mechanisms of current treatments, but is also important for the identification of new targets for biological intervention. An IgE-specific monoclonal antibody, omalizumab, has already reached the clinic and similar biological agents will surely follow. Key words: bronchial asthma, cytokines, dendritic cells, IgE, Th2 lymphocytes, corticosteroids, anti-IgE monoclonal antibodies. Corresponding author: Prof. Milan Buc, MD, Ph.D., Department of Immunology, Comenius University School of Medicine, Bratislava, Slovakia, tel.: +421 2-59357398, fax: +421 2-59357578, e-mail:
[email protected]
Bronchial asthma is a chronic inflammatory disease characterized by a variable degree of airflow obstruction occurring spontaneously or in response to nonspecific triggers such as exercise, smoke, and fumes. Most patients have mild disease, but a significant minority suffers from more severe forms of the disease despite optimal medical management. There are basically two forms of asthma, allergic and non-allergic (intrinsic). The immunopathology of non-allergic asthma appears to be very similar to that of allergic asthma, although there are some differences (Humbert et al. 1999). Allergic asthma is a disease derived from airway inflammation and bronchoconstriction, which is, however, a secondary phenomenon of the disease (1991). In developed countries, 30% of the population is atopic, but only 10–12% of the population actually suffers from asthma (Hammad and Lambrecht 2008). It is therefore crucial to understand the genetic and environmental factors as well as pathogenic mechanisms leading to allergic inflammation that all eventually result in bronchial asthma development. Much progress in our knowledge of atopic reactions has been made in recent years and the
aim of this article is to offer the reader a review of these achievements and their impacts on immunotherapy.
UPTAKE AND TRANSPORT OF INHALED ALLERGENS Under physiological circumstances, the epithelium forms a highly regulated and almost impermeable barrier through the formation of tight junctions. The epithelial cell layer acts as a molecular sieve that excludes inhaled antigens and pathogens. However, some antigens can be recognized by cells of the immune system and induce an immune response. Mucosal dendritic cells (DCs) are extremely efficient sentinels in the defense against antigen challenge. They are strategically positioned within the epithelium in the basolateral space, separated from the inhaled air only by the epithelium tight junction barrier. However, DCs extend their processes between epithelial cells directly into the airway lumen (Fig. 1). This “periscope” function provides a mechanism for contin-
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Fig. 1. Periscope function of DCs (modified from Hammad and Lambrecht 2008). DCs intercalate their dendrites between epithelial cells, which enables them to sample the airway lumen. DCs form tight junctions with epithelial cells through their expression of adhesive molecules and by E-cadherin homotypic interactions.
uous immune surveillance of the airway luminal surface (Jahnsen et al. 2006). Although the sampling function of airway DCs ensures that virtually any inhaled protein will be recognized and presented to T cells following inhalation, some allergens, for example those from house dust mites, compromise the epithelial barrier function by degrading tight junction proteins, thus facilitating allergen delivery across epithelial layers (Runswick et al. 2007; Shakib et al. 2008). Other allergens that lack protease activity, such as cockroach-derived allergens, can increase the permeability of bronchial airway epithelial cells indirectly through the induction of vascular endothelial growth factor (VEGF) (Antony et al. 2002). Despite the fact that most inhaled antigens are transported to the lymph nodes by DCs, the usual outcome following the inhalation of harmless protein antigens is the induction of tolerance. This is because they cannot fully activate DCs to induce an effective T-cell response (Reis e Sousa 2006). It follows that DCs have to be somehow activated to break tolerance. Conventional DCs express numerous pattern-recognition receptors, including Toll-like receptors (TLRs), nucleotide-binding oligomerization domain and C-type lectin receptors (Sandor and Buc 2005a; Buc 2005b; Buc 2005c; Suarez et al. 2008). As most inhaled allergens, such as those derived from cockroaches and house-dust mites, are contaminated with lipopolysaccharides (LPSs) and peptidoglycans, they can activate DCs. In fact it was shown that the main house dust mite allergen, Der p 2, acted as a functional homologue of myeloid differentiation factor 2 (MyD2) that drove airway inflammation in a TLR4-dependent manner (Trompette et al. 2009). MyD2 physically associates with the extracellular domain of human TLR4 and binds the lipid A region of LPS without the need for LPS-binding protein (Viriyakosol et al. 2000). Given that many allergens, including Der p 2, are members of the MyD2-like lipid-binding protein family and that more than 50% of
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major allergens are lipid-binding proteins, such mimicry could also explain the immunogenicity of these allergens. In the absence of contaminating TLR ligands, some allergens can activate DCs by triggering protease-activated receptors (PARs). They are present on many cell types, including epithelial cells, fibroblasts, smooth muscle cells, endothelial cells, and on a number of inflammatory cells. There is increasing evidence that besides DC activation, they are also involved in mesenchymal cell proliferation and airway wall remodeling (Berger et al. 2001). The ligation of TLRs and PARs leads to a cascade of events that culminates in the production of chemokines that attract neutrophils, monocytes, and DCs to the airways and to the production of cytokines that can induce DC maturation and Th2 polarization (Kiss et al. 2007). Thymic stromal lymphopoietin (TSLP), granulocytemonocyte colony-stimulating factor (GM-CSF), and interleukin (IL)-25 are among the most important. TSLP is a cytokine with a strong capacity to modulate an immune response. TSLP-stimulated DCs are able to prime human CD4+ T cells into Th2 cytokine-producing cells (Ito et al. 2005; Wang et al. 2006). Moreover, it was shown that TSLP was able to prime Th2 differentiation directly, in the absence of DCs; the treatment of naïve CD4+ T cells with TSLP in the presence of T cell receptor (TCR) stimulation led to IL-4 production and their differentiation into the Th2 subset (Omori and Ziegler 2007). In addition to its effects on DCs and naïve CD4+ T cells, TSLP can also activate mast cells to produce Th2-cell associated effector cytokines (Holgate 2008). GM-CSF is another important cytokine that is produced by airway epithelial cells in response to proteolytic allergen exposure. Known Th2-cell sensitizers such as diesel exhaust particles, ambient particulate matter, and cigarette smoke also induce the release of GM-CSF from epithelial cells (Bleck et al. 2006). GM-CSF stimulates the proliferation and differentiation of precursors of neutrophils, eosinophils, and monocytes. It also functionally activates the corresponding mature forms, enhancing, for example, the expression of certain cell-surface adhesion proteins (LFA-1, Mac-1). The over-expression of these proteins could be one explanation for the observed local accumulation of neutrophils and eosinophils at sites of inflammation. Moreover, GM-CSF stimulates in them the release of arachidonic acid metabolites and the increased generation of reactive oxygen intermediates (ROI) (Holgate 2008). Antigen uptake and TSLP trigger the maturation of DCs and their migration to mediastinal lymph nodes, during which they up-regulate the expression of HLA class II antigens and co-stimulatory molecules. When they enter the lymph nodes, the already mature DCs induce the activation and polarization of naïve T-helper cells. This process involves interactions between the co-stimulatory molecules OX40 (CD134) in the membranes of naïve T cells and OX40L (CD134L) in the membranes of DCs. The co-stimulatory interactions result in the
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Fig. 2. Polarization of naïve helper T cells to inflammatory Th2 subset. Allergens trigger mucosal epithelial cells to produce TSLP. TSLP activates immature DCs. TSLP-activated DCs express OX40L, which interacts with OX40 (CD134) in the membranes of naïve T cells. DC-activated Th cells (aTh) start to produce IL-4 and to differentiate to inflammatory Th2 cells (iTh2) that produce IL-4, IL-5, IL-13, and TNF, but not IL-10. The Th2 cytokines IL-4, IL-5, IL-13, and TNF produced by the inflammatory Th2 cells initiate allergic inflammation by triggering IgE production, eosinophilia, and mucus production.
induction of IL-4 production by T cells followed their subsequent polarization into the Th2 subset. The activated allergen-specific Th2 cells synthesize IL-4, IL-5, IL-13, and tumor necrosis factor (TNF)-α, but not IL-10 (inflammatory Th2 cells1) (Hammad and Lambrecht 2008; Ito et al. 2005) (Fig. 2). The polarization of Th2 cells induced by TSLP-matured DCs is further enhanced by IL-25 that is produced by epithelial cells, basophils, and eosinophils. IL-25 co-stimulates the proliferation of Th2 cells and cytokine production, in particular IL-5. The finding that IL-25 receptor is highly expressed on CD4+ Th2 cells in humans provided direct evidence that IL-25 can directly act on CD4+ T cells, thus potentiating the Th2-type immune response (Wang et al. 2007). Finally, increased burden of inhaled oxidants and increased amounts of reactive oxygen species generated by several inflammatory, immune, and structural cells of the airways either directly or via the formation of lipid peroxidation products play a role in Th2-cell development (Rahman 2002). Experimental evidence in recent years shows that a significant portion of already known allergens has an intrinsic protease activity. However, secreted proteases 1 Th2 cells are usually defined as CD4+ T cells that produce IL-4, IL-5, IL-13, and IL-10. When TSLP-DCs are used to stimulate naïve allogeneic CD4+ T cells in vitro, they induce a unique type of Th2 cell that produces the classical Th2 cytokines IL-4, IL-5, and IL-13 and large amounts of TNF, but little or no IL-10. Such Th2 cells were proposed to be called inflammatory in contrast to conventional Th2 cells.
are essential for the infectious and reproductive cycles of helminths. It is therefore possible that the innate immune system has evolved a detection mechanism based on sensing protease activity associated with helminth infection. The ability of protease allergens to trigger the Th2 response could then be explained by their ability to trigger the same pathway that has evolved to fight helminth infections (Finkelman and Urban 1992; Sokol et al. 2008).
CELLS AND MOLECULES IN ALLERGIC INFLAMMATION A basic feature of allergen sensitization is the uptake and processing of inhaled allergens by DCs. Once inside DCs, the processing of allergens and the subsequent loading of emerging peptides into the grooves of HLA class II molecules is fundamental to the ability of DCs to serve as antigen-presenting cells (Hintzen et al. 2006; Neefjes 1996). By allergen processing, DCs migrate to local lymph nodes to present and activate naïve T cells. Their specific chemokine receptors, mainly CCR7, and their ligands, CCL19 and CCL21, are involved in this chemotactic migration (Burgess et al. 2005; Wang et al. 2006). Recognition of the presented peptides by the TCR and co-stimulatory interactions between DCs and T cells initiate the activation of T cells with subsequent polarization (see above). Dendritic cells are not only involved in the initial sensitization to allergens, but also in the recruitment of
334 Th2 cells to the airways. TSLP released by activated epithelial cells induces DCs to produce chemokines CCL17 (TARC) and CCL22 (MDC), which bind to CCR4 that is selectively expressed by Th2 cells. It is this type of T cell which dominates the allergic immune response and may be the cell most probably responsible for contributing to the ongoing chronic inflammatory response (Barnes 2008a; Holgate 2008; Liu 2006). Thus CCL17 and CCL22 attract Th2 cells to the site of allergic inflammation by means of a chemical gradient. Cigarette smoking substantially contributes to this process; it was shown that α-glycoprotein, isolated from tobacco, substantially increased the number of mature DCs in the airways and alveolar walls (Soler et al. 1989). Th2 cells have a central role in allergic inflammation. When coordinately stimulated, they up-regulate the expression of a cluster of genes encoded on chromosome 5q31–33 which includes the genes encoding IL-3, IL-4, IL-5, IL-9, IL-13, and GM-CSF. These cytokines are involved in the class-switching of B cells to IgE synthesis (IL-4 and IL-13), the recruitment of mast cells (IL-4, IL-9, and IL-13), and the maturation of eosinophils (IL-3, IL-5, and GM-CSF) and basophils (IL-3 and IL-4), the main mediator-secreting effector cells of the allergic response (Barnes 2008a; Kay 2006). The transcription factor GATA3 is crucial for the differentiation of uncommitted naïve T cells into the Th2 subset and also regulates the secretion of Th2-type cytokines. GATA3 expression is controlled by signal transducer and activator of transcription 6 (STAT6), which is induced by IL-4 binding to its receptor (Barnes 2008b). The crucial transcription factor for Th1 cell differentiation is T-BET. Consistent with the prominent role of Th2 cells in asthma, T-BET expression is reduced in T cells from the airways of asthmatic patients compared with non-asthmatic subjects. When phosphorylated, T-BET can associate with and inhibit the function of GATA3 by preventing it from binding to its DNA target sequences. GATA3 expression is also regulated by IL-27, which down-regulates GATA3 and up-regulates T-BET, thereby favoring the production Th1-type cytokines (Barnes 2008a). Finally, it was recently shown that IL-33 in the presence of antigen, by binding the surface receptor ST2, polarized naïve CD4+ T cells into a population of T cells which produce IL-5 and IL-13, but not IL-4. This polarization requires IL-1R-related molecule and MyD88, but not IL-4 or STAT6. The IL-33-induced T-cell differentiation is also dependent on the phosphorylation of mitogen-activated protein kinase and NF-κB, but not the induction of GATA3 or T-BET (Kurowska-Stolarska et al. 2008). This new T-cell subpopulation, Th5, thus promotes airway inflammation independent of IL-4. Moreover, IL-33 also acts as a selective chemoattractant of Th2 cells (Komai-Koma et al. 2007). Whereas Th2 cells predominate in mild asthma, in more severe forms of asthma both Th1 and Th2 cells as well as the more cytotoxic CD8+ T cells are present in bronchial biopsies (Barnes 2008a).
M. Buc et al.: Immunopathogenesis of bronchial asthma
Other subtypes of CD4+ T cells that may have an important role in allergy development are regulatory T lymphocytes, which have a suppressive effect on other CD4+ T cells. There is evidence that the number of CD4+CD25+FOXP3+ regulatory T cells (Tregs) is reduced in individuals with allergic rhinitis compared with non-atopic individuals. However, by contrast, asthmatic patients seem to have an increase in Tregs compared with patients with mild asthma, at least among circulating cells (Lee et al. 2007). The role of Tregs in asthma therefore remains unclear and further research is needed. The Th17 subset of CD4+ T cells has recently been shown to have an important role in inflammatory and autoimmune diseases (Stockinger and Veldhoen 2007; Weaver et al. 2006). Little is known about the role of Th17 cells in asthma, but increased concentrations of IL-17 (the principal cytokine produced by Th17 cells) have been reported in the lungs, sputum, and bronchoalveolar lavage fluid of asthma patients. The severity of airway hypersensitivity in patients correlates with the level of IL-17 expression, suggesting that IL-17 cytokines play an important role in driving allergic inflammation (Bullens et al. 2006). IL-17 can orchestrate local inflammation by inducing the release of proinflammatory cytokines such as TNF-α, IL-1β, G-CSF, and IL-6 as well as chemokine CXCL1 (Gro-α) and CXCL8 (IL-8) production by bronchial fibroblasts and epithelial and airway smooth muscle cells. Furthermore, IL-17 can act in synergy with IL-6 to induce mucus proteins (MUC5B and MUC5AC) or with IL-1β and TNF-α to enhance VEGF (Wang and Liu 2008). Chemokines CXCL1 and CXCL8 act on CXCR2, expressed predominantly by neutrophils, resulting in neutrophilic inflammation, especially in severe asthma (Barnes 2008a; Keatings et al. 1996). Th17 cells also produce IL-21, which supports allergic inflammation by inhibition of FOXP3 expression and Treg lymphocyte development (Spolski and Leonard 2008). However, more work is needed to understand the role of Th17 cells in asthma. Although allergic diseases have clearly been associated with cytokines produced by Th2 cells, the source of these Th2 cytokines can also differ. Among other cells, invariant TCR+ CD1d-restricted CD4+ natural killer T (NKT) cells can also be taken into account as they secrete IL-4 and IL-13 (Jinquan et al. 2006). The exact mechanisms by which NKT cells are activated during Th2 immune responses are currently unknown; there are principally two possibilities. First, allergens may contain glycolipids that are recognized by the invariant TCR of NKT cells. Second, self-glycolipid antigens may be released and recognized during the course of inflammatory responses. NKT cells have been shown to account for 60% of all CD4+ T lymphocytes in bronchial biopsies from asthmatic patients (Akbari et al. 2006), but this was disputed in another study (Vijayanand et al. 2007), so the role of NKT cells in asthma is currently uncertain and further studies will be required to deter-
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mine the extent of the contribution of NKT cells in various allergic disorders and to determine the specific endogenous or exogenous glycolipid antigens that activate NKT cells in allergic patients. B cells have an important role in allergic diseases, including asthma, through the synthesis of allergen-specific IgE. IgE binds to its high- (FcεRI) and low-affinity receptors (FcεRII). All granulocytes have been reported to express FcεRI; monocytes, DCs, and Langerhans cells also express modest levels of FcεRI. Beyond the hematopoietic cells, there are some reports of smooth muscle and nerve cells expressing FcεRI; many of these cell types do not express the beta subunit (MacGlashan 2008; Rivera et al. 2008). Extracellular domains of the α-chain are responsible for IgE binding, whereas the signaling motifs (immunoreceptor tyrosine-based activation motifs) are located in the intracellular parts of the β- and γ-chains (MacGlashan 2008; Rivera et al. 2008). FcεRII (CD23) is a calcium-dependent type II integral membrane protein that belongs to the lectin family of adhesion molecules. FcεRII exists in two forms, CD23a and CD23b, resulting from alternative splicing at the N-terminus. Both isoforms of CD23 are found on B cells; one, CD23a, is constitutively expressed, whereas the other form, CD23b, is induced by factors such as IL-4 and CD40L in conjunction with IL-4. CD23b is also found on non-B cells such as T cells, Langerhans cells, monocytes, macrophages, platelets, and eosinophils (Gould and Sutton 2008). Structurally, CD23 presents a single membrane-spanning domain followed by an extracellular domain that consists of three regions: the α-helical coiled-coil stalk region, which mediates the formation of trimers, followed by the lectin head, which binds IgE, and at the C-terminus a short tail containing a common recognition site of integrins (Fig. 3). The binding of IgE allows membrane CD23 to deliver a negative IgE regulatory signal to the B cell which makes it stop further IgE synthesis (Lamers and Yu 1995). The stalk region is susceptible to proteolysis, leading to the release of soluble fragments. The principal endogenous protease that releases the soluble CD23 is a disintegrin and metalloproteinase 10 (ADAM10) (Weskamp et al. 2006). The stalk region has also been implicated in binding to HLA class II molecules (Hibbert et al. 2005). Surprisingly for a lectin, the ability of CD23 to bind IgE does not involve binding to carbohydrate. However, CD23 binds in a carbohydrate-dependent manner its second ligand, CD21 (Aubry et al. 1994). It is thought that soluble CD23 will stimulate an up-regulation of IgE synthesis by the co-ligation of membrane IgE and CD21. This mechanism is analogous to that seen in the co-ligation of the IgM-receptor of B cells and CD21 (CR2, complement receptor 2) by fragments of complement component C3 (C3d) attached to an antigen which stimulates the immune response (Aubry et al. 1994; Gould and Sutton 2008; Montagnac et al. 2005). As CD21 is expressed by B cells, follicular DCs, activated T cells, and basophils, adhesion pairing between these two mol-
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Fig. 3. The structure of CD23 (modified from Gould and Sutton 2008). CD23 is a transmembrane glycoprotein. Its extracellular portion consists of three lectin domains that bind IgE and a trimeric α-helical coiled-coil “stalk”. N-linked glycosylation sites are near the base of the stalk.
ecules may have important consequences for allergy. For example, Der p 1, the major house dust mite antigen, has been shown to selectively cleave CD23 that could promote IgE synthesis (Shakib et al. 1998). Soluble CD23 also ligates CD11b/CD18 (Mac-1) and CD11c/CD18 (CR4) molecules, thus promoting the release of pro-inflammatory mediators such as IL-1β, IL-6, and TNF-α. Allergen-IgE complexes bound in the mucosa to CD23 that is expressed by allergen-activated B cells can facilitate antigen presentation to T cells. The process of antigen presentation by means of CD23 is termed facilitated antigen presentation (Gould and Sutton 2008). The interaction between CD23 and HLA-DR in the cell membrane is involved in the trafficking of allergen-IgE-CD23 complexes to endosomes, where the allergen-derived peptides are loaded into the grooves of HLA-DR molecules for presentation (Karagiannis et al. 2001). CD23-expressing B cells are so able to present a variety of peptides, even from totally unrelated allergens, to T cells. This is a probable scenario for “epitope spreading” (Mudde et al. 1995). IgE and FcεRI can also be bound by galectin 3. This pentameric molecule, by cross-linking receptor-bound IgE, FcεRI, or both, can activate mast cells or basophils. Its expression is elevated in peribronchial macrophages and it is released into the extracellular space, where it contributes to activation and to the retention of cells. Through binding to FcεRI, galectin 3 may itself contribute to airway smooth muscle-cell contractile responses and thus to airway hyperresponsive-
336 ness and remodeling in asthma (Gould and Sutton 2008; Liu 2005). Mast cells have a key role in asthma. They are concentrated in the mucosal tissues and are recruited to the surface of the airways by stem-cell factor released from epithelial cells. In addition, CXCL8 and CXCL10, produced by airway smooth muscle cells, are important in the recruitment of mast cells by interacting with their receptors, CXCR2 and CXCR3, respectively. Moreover, these chemokines also prime mast cells for enhanced mediator secretion. Reversely, mast cells secrete CCL19 which, through its CCR7, stimulates airway smooth muscle cell migration and contributes to smooth muscle hyperplasia (Holgate 2008; Kaur et al. 2006). The cross-linking of IgE-FcεRI complexes on mast cell surfaces by allergens leads, within minutes, to the so-called “early phase” of the allergic reaction, which involves their degranulation and release of histamine, tryptase and other proteases, heparin and some cytokines, which are preformed and stored in granules, as well as newly formed eicosanoids (LTC4, LTD4, LTE4, PG2, and TXA2). These mediators are potent smooth muscle contractile agents and also increase microvascular permeability. Both PGD2 and LTD4 interact with cell-surface receptors on eosinophils, macrophages, basophils, and mast cells, where they serve as chemoattractant as well as priming agents. The release of mediators may account for the bronchoconstriction seen in asthma, as these mediators are released by various environmental triggers. Cytokines and chemokines liberated in this early phase initiate the “late phase”, which peaks some hours later (Gould and Sutton 2008). Mast cells and eosinophils are also an important source of the zinc-dependent matrix metalloproteinases 3 and 9 (MMP-3, MMP-9) which, through their interaction with matrix proteins and proteoglycans, have also been incriminated in airway wall remodeling (Wenzel et al. 2003). Basophils are rare circulating granulocytes that originate from CD34+ hematopoietic progenitors in the bone marrow. They are believed to complete their maturation there and thus exit the bone marrow fully matured. Whereas mast cells persist in tissues, basophils are rarely found there. Basophils are short-lived cells with an expected half-life of approximately a few days. The number of basophils increases considerably in sites of allergic inflammation and they substantially contribute to its development. In fact, basophils resemble mast cells in many ways; both cells express FcεRI and both rapidly produce cytokines, histamine, and lipid mediators after cross-linking of their FcεRI receptors (Min 2008; Min and Paul 2008). However, it seems that basophils are the main cells that produce IL-4 and IL-13 after allergen challenge and thus promote the development of the Th2-biased immune response. Moreover, it has been shown that, after IgE cross-linking, basophils also secrete IL-25, for which Th2 memory cells express the specific receptor in high quantities (Wang et al. 2007). Basophils have a high expression of IL-33 recep-
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tor and produce IL-4 and IL-13 in response to this cytokine (Kondo et al. 2008). Activated basophils were shown to produce TSLP as well (Min and Paul 2008; Sokol et al. 2008). Basophils have also been shown to induce switching of the B-cell isotype to IgE in vitro. Activated basophils up-regulate the surface expression of CD40 ligand, which interacts with CD40 on B cells to provide help for their differentiation. Blockade of CD40L is sufficient to abrogate IgE synthesis, which further supports the idea of a critical involvement of the CD40 ligand-CD40 interaction in this process (Yanagihara et al. 1998). The inflammation that occurs in asthma is often described as eosinophilic. Eosinophils are present not only in the airway wall, but in uncontrolled asthma also in the sputum and bronchoalveolar lavage fluid (Holgate 2008). These cells are in large part initially recruited from the bone marrow as precursors following the release of PGD2, leukotrienes, and cytokines and chemokines (IL-3, GM-CSF, eotaxins 1, 2, and 3, RANTES) (Sehmi et al. 2003). Eosinophils are a rich source of granule basic proteins, such as major basic protein, eosinophil peroxidase, and eosinophil cationic protein, and also have the capacity to generate eicosanoids such as prostacyclin (PGI2) and leukotrienes. They also release potentially tissue-damaging superoxide and a range of cytokines and chemokines. Eosinophil-derived neurotoxin is released by eosinophils as well. It was recently shown that it had the capacity to activate Th2-polarizing DCs by triggering the TLR2-MyD88 signaling pathway and to enhance the Th2-biased immune response (Yang et al. 2008). Eosinophils, by their capacity to generate TGF-β and support of fibroblast proliferation, collagen synthesis, and myofibroblast maturation, also contribute to tissue remodeling (Williams and Jose 2000). Some patients with asthma have neutrophils instead of eosinophils in their sputum. In general, asthma associated with neutrophils tends to be more severe, probably because of increased tissue destruction and airway remodeling (Holgate and Polosa 2006) (see later). Macrophage numbers are increased in the lungs of patients with asthma. They are derived from circulating monocytes, which migrate to the lungs in response to chemoattractants such as CCL2 (MCP1), acting on CCR2, and to CXCL1 (GROα), acting on CXCR2 (Sandor and Buc 2005c). Although these cells are an important source of leukotrienes, ROI, and a variety of lysosomal enzymes, their precise role in mediating tissue damage and contributing to the overall airway pathology in asthma is largely unknown. In corticosteroid-refractory asthma, monocytes and macrophages are thought to play an increasingly important role (Loke et al. 2006).
THE AIRWAY EPITHELIUM IN ASTHMA AND REMODELING The airway epithelium, while playing an important role as a physical barrier, is now recognized to be fun-
M. Buc et al.: Immunopathogenesis of bronchial asthma
damental to asthma pathogenesis. Moreover, it undergoes structural changes, referred to as airway remodeling. Bronchial biopsies from asthmatic subjects reveal infiltration of eosinophils, activated mast cells, and activated T cells. There are characteristic structural changes with collagen deposition under the epithelium, described as basement-membrane thickening and thickening of the airway smooth-muscle cell layer as a result of hyperplasia and hypertrophy. The process is accompanied by an increased number of mucus-secreting goblet cells in the epithelium, enlargement of submucosal glands, and raised number of blood vessels due to aggravated secretion of VEGF. This indicates that the epithelium is chronically injured and unable to repair properly, mostly in patients suffering from severe asthma (Barnes 2008a; Holgate 2008, Wang et al. 2006). Under normal circumstances the epithelium forms a highly regulated and almost impermeable barrier through the formation of tight junctions. It is now clear that in asthma the epithelium is more fragile, with easy loss of columnar cells due to disruption of both tight junctions and desmosomal attachments (Barbato et al. 2006). The permeability of the asthmatic epithelium is greatly increased, leading to greater access of inhaled allergens, pollutants, and other irritants to basal cells and the underlying airway tissue. This loss of barrier function may reflect a broader abnormality; the association of genetic polymorphism of the profilaggrin gene on chromosome 1q13 with atopic dermatitis and asthma was described (Ying et al. 2006). Filaggrin and the S100 proteins are involved in maintaining epithelial integrity in both the skin and the airways. Another possibility of an environmental injury targeting the airway epithelium is the effect of respiratory viral infections. It has long been known that common cold viruses, such as rhinoviruses, are associated with exacerbations of asthma. The airway epithelium is the preferential site for these viruses to enter the airway tissue. Viral infection of normal epithelial cells generates sufficient quantities of type I interferons that are essential for the elimination of viruses. Asthmatic epithelium, however, loses this ability and viruses continue to replicate until they kill the epithelial cells, leading to massive virus shedding and infection of adjacent cells as well as the release of mediators from damaged cells (Wark et al. 2005). Air pollution contributes to asthma worsening as well. Many studies have shown that the antioxidant defense mounted by the airway epithelium in asthma is markedly reduced and is associated with a reduction in superoxide dismutase (Comhair et al. 2005). In such a way the airway epithelium is damaged more easily as it is not able to defend itself adequately against oxidant damage. The association of asthma with ozone and particle pollution episodes can be explained in this way. When damaged, the airway epithelium needs to repair. However, the repair process is compromised in asthma, which results in the production of a variety of cytokines and growth factors in an attempt to repair
337 damaged cells. Epidermal growth factor stimulation of the damaged epithelium generates a mucus-secretory phenotype and an altered inflammatory response involving neutrophils, GM-CSF and chemokines attract neutrophils and other inflammatory cells, and platelet-derived growth factor, fibroblast growth factor (FGF)-1, FGF-2, and TGF-β are active on fibroblasts and smooth muscle (Holgate 2008; Casalino-Matsuda et al. 2006; Hamilton et al. 2003). The results of their cumulative activities are the morphopathogenic changes so characteristic of asthma, especially the more chronic and severe forms. The airway epithelium is stratified; the upper layer contains a mixture of ciliated, goblet, and Clara cells. In chronic asthma the number of goblet cells, which secrete viscous mucus, increases, with a parallel reduction of ciliated cells. This metaplastic change is of great importance as it occurs in the more peripheral airways, which are normally devoid of goblet cells (Perez-Vilar 2007). The factors responsible for goblet cell metaplasia include IL-4, IL-9, IL-13, and TNF-α, which are all capable of directing differentiation to a mucus-secreting phenotype (Holgate 2008). Recently, IL-13 was shown to regulate a chloride channel which is intimately involved in the regulation and secretion of mucin from goblet cells (Thai et al. 2005). The airways become thickened in chronic asthma. Bronchial biopsies from asthma patients have revealed that myofibroblasts are present in increased numbers in the subepithelial and submucosal region of asthmatic patients and increase in proportion to disease chronicity and severity. However, the airways also become thickened as a consequence of the deposition of proteoglycans, with their ability to sequestrate water (Barnes 2008a; Wicks et al. 2006). Paradoxically, airway wall thickness is inversely correlated with airway hyperresponsiveness, suggesting that this thickening with deposition of matrix proteins may be a protective response against frequent smooth muscle contraction (Paganin et al. 1996). In the process of remodeling, one enzyme is of paramount importance, i.e. ADAM33. It is preferentially expressed in airway mesenchymal cells. Although the full-length molecule is expressed as a transmembrane protein, a soluble form exists as well; more importantly, its levels increase in proportion to disease severity (Foley et al. 2007). Of the many potential biological actions that ADAM33 possesses, its proteolytic activity, which is also present in the soluble form, is likely to be important in generating growth factors that influence mesenchymal cell number and/or maturation (Holgate 2008; Holgate et al. 2006). Together, these observations point to fact that the airway epithelial and mesenchymal cells are abnormal in both their response to environmental injury and its repair and induce immunopathological reactions in genetically susceptible individuals. This abnormality may form a basis for the onset of asthma; the altered microenvironment in the airway may provide an oppor-
338 tunity for inhaled allergens, pollutants, and viruses to gain access and induce the characteristic immunopathological and inflammatory processes.
HETEROGENEITY OF ASTHMA Bronchial asthma is not a single disease, but rather a complex of multiple separate syndromes that overlap. Although clinicians have recognized these different phenotypes for many years, they have remained poorly characterized. However, the recent understanding of immunopathological processes enabled distinguishing at least four distinct phenotypes or endotypes: eosinophilic, neutrophilic, paucigranulocytic, and steroid-resistant asthma (Anderson 2008; Holgate 2008; Simpson et al. 2006; Wenzel 2006). Eosinophilic asthma Eosinophilic airway inflammation is highly characteristic for patients with mild asthma. A broad correlation between clinical asthma severity and the degree of airway eosinophilia has been recorded (Bochner and Busse 2005). Moreover, the dramatic reduction of eosinophils in sputum and tissue as a result of asthma treatment with corticosteroids, associated with clinical improvement, has led to the notion that eosinophils are fundamental to airway dysfunction in asthma. However, the role of eosinophils has been recently questioned, as the administration of an anti-IL-5 monoclonal antibody, mepolizumab, that reduces their number in the blood and sputum, does not reduce airway hyperresponsiveness or asthma symptoms (Leckie et al. 2000). Moreover, anti-IL-5 treatment had no effect on bronchial mucosal staining of eosinophil major basic protein, suggesting that reduction in eosinophil numbers does not reflect the tissue deposition of granule proteins. Therefore, tissue eosinophils may be unresponsive to IL-5, but may instead respond to IL-3 and GM-CSF (Flood-Page et al. 2007; Flood-Page et al. 2003). In any case, eosinophils may be responsible for subepithelial fibrosis, and their presence in the airways seems to be a good marker of steroid responsiveness Also, eosinophilic airway inflammation appears to be much more closely related to the risk of severe asthma exacerbation (Green et al. 2002). Neutrophilic asthma Some patients with asthma have neutrophils in their sputum instead of eosinophils, as expected. In general, asthma associated with neutrophils tends to be a more aggressive disease, with more tissue destruction and airway remodeling (Holgate and Polosa 2008; Wenzel 2006). Intervention with etanercept, a p75 TNF-α receptor fusion protein (TNF-α is a potent chemoattractant for neutrophils), has shown clinical benefit in such patients (Barnes 2008a). This suggests that as the
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disease becomes more chronic and severe, the inflammatory phenotype changes from a Th2 more towards a Th17 type, as Th17 cells, by their IL-17 production, induce the release of CXCL8 (IL-8), a neutrophilic chemokine, from airway epithelial cells (Wang and Liu 2008). Neutrophilic asthma has also been associated with infections. Since asthmatic airway epithelial cells are known to be deficient in their ability to mount a primary interferon response following infection with common respiratory viruses (Wark et al. 2005), it is possible that defective innate immunity may be fundamental to the origin and progression of chronic disease. A neutrophilic pattern of inflammation is also found in asthmatic patients who smoke. It was shown that α-glycoprotein, isolated from tobacco, substantially increased the number of mature DCs in the airways and alveolar walls (Soler et al. 1989) and that cigarette smoke induced high levels of CXCL8. Tobacco smoking is not only associated with a greater neutrophil component, but also, and more importantly, with corticosteroid refractoriness (Livingston et al. 2007). A possible explanation for this is the effect of smoking and oxidative stress in reducing histonedeacetylase activity (see below) in the nuclear chromatin, thereby diminishing the opportunity for corticosteroids to access anti-inflammatory genes (Adcock et al. 2008). Paucigranulocytic asthma Asthma in the absence of either neutrophils or eosinophils and normal levels of proteolytic enzymes in the patient’s sputum is termed paucigranulocytic. Proteolytic enzymes play an important role in tissue remodeling and repair, but their levels and activity vary according to the inflammatory cell phenotype. Subjects with eosinophilic asthma have significantly more active MMP-9 compared with those with neutrophilic asthma and control subjects. In neutrophilic asthma, more subjects have neutrophil elastase (NE) activity compared with healthy control subjects, subjects with eosinophilic asthma, or subjects with paucigranulocytic asthma (Simpson et al. 2005). Proteolytic enzyme activity in asthma is thus dependent on the underlying inflammatory phenotype and is differentially regulated, with MMP-9 activity a feature of eosinophilic inflammation and active NE in neutrophilic inflammation. The normal levels of MMP-9 and NE in paucigranulocytic asthma suggest that an abnormal epithelium or underlying mesenchyme and/or smooth muscle may itself lead to an asthma phenotype without the presence of obvious inflammation (Holgate 2008). Steroid-resistant asthma Glucocorticoids are in the first-line of anti-inflammatory treatment for asthma. However, a proportion of asthmatic patients fails to benefit from oral glucocorticoid therapy; they are denoted as having glucocorticoid-
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-resistant (steroid-resistant) asthma. The molecular and cellular basis of steroid resistance remains uncertain. Some investigations have shown that steroid insensitivity in these patients is associated with a breakdown of nuclear translocation of the glucocorticoid receptor (Goleva et al. 2006). Some patients who have clinically severe asthma despite taking oral and high-dose inhaled steroids show persistent airway neutrophilia and increased expression of both TNF-α mRNA and protein (Berry et al. 2006). It was also shown that CD4+ T cells from steroid-resistant asthma patients failed to induce IL-10 synthesis following in vitro stimulation in the presence of dexamethasone compared with their glucocorticoid-sensitive counterparts, suggesting a link between the induction of IL-10 synthesis and the clinical efficacy of glucocorticoids (Xystrakis et al. 2006).
PHARMACOTHERAPY Inhaled corticosteroids and short- and long-acting β2-adrenoceptor agonists are now the mainstay of asthma treatment (Barnes 2006a; Holgate 2008; Holgate and Polosa 2008). Corticosteroids suppress inflammation by inducing the recruitment of the nuclear enzyme histone deacetylase 2 (HDAC2) to multiple activated inflammatory genes, which leads to deacetylation of the hyperacetylated genes, thereby suppressing inflammation. The poor response to corticosteroid treatment seen in patients with severe asthma, in asthmatics who smoke, and during acute exacerbations may also reflect a reduction in HDAC2 enzyme levels and its function. Inhaled steroids also reduce the number of DCs in the lungs and activate indolamine 2,3-dioxygenase in plasmacytoid DCs, thereby broadly suppressing pro-inflammatory responses (Barnes 2006b; Barnes 2008a). Corticosteroids can dampen airway inflammation, although they have little effect on remodeling. This explains why corticosteroids do not abolish all symptoms (Barnes 2006a; Holgate 2008; Holgate and Polosa 2008). While antihistamines can block the effects of allergen inhalation, these drugs show little benefit in clinical asthma, suggesting that histamine is not an important mediator, except perhaps in acute bronchospasm induced by heavy allergen exposure, for example in patients who wheeze on acute exposure to cats. In contrast, leukotriene antagonists (LTRAs) are clinically effective, confirming that leukotrienes are relevant mediators of asthma. They induce bronchodilation and inhibit airway constriction induced by antigen inhalation or exercise and also exert an anti-inflammatory effect (Pizzichini et al. 1999). Furthermore, because leukotrienes are known to play an important role in an airway remodeling, LTRAs can be used as its inhibitors (Lex et al. 2006). Cyclosporine A was found to be of little benefit to asthmatics and is now not recommended as a therapy, particularly because of its toxicity. Tacrolimus, rapamycin, and mycophenolate mofetil, which are cur-
rently used in the prevention of transplant rejection, have not been tested in clinical studies of asthma (Evans et al. 2001). FTY720, a sphingosine 1 phosphate receptor antagonist, is an immunosuppressant that retains lymphocytes in lymph nodes and spleen, thus preventing lymphocyte migration to inflammatory sites (Cahalan and Parker 2006). It has currently been used in clinical trials for the treatment of multiple sclerosis and transplant rejection. The accompanying lymphopenia could be a serious side effect that would preclude the use of FTY720 as an antiasthmatic drug. However, it was shown that the administration of FTY720 by inhalation prior to or during ongoing allergen challenge suppressed Th2-dependent eosinophilic airway inflammation and bronchial hyperresponsiveness in mice without causing lymphopenia and T-cell retention in the lymph nodes. The effectiveness of local treatment was achieved by inhibition of the migration of lung DCs to the mediastinal lymph nodes, which in turn inhibited the formation of allergen-specific Th2 cells in lymph nodes (Idzko et al. 2006). However, more studies are needed before the drug enters clinical practice. Theophylline has been used to treat asthmatic bronchoconstriction. It is a cAMP phosphodiesterase inhibitor as well as an adenosine-receptor antagonist. Theophylline is reported to have anti-inflammatory efficacy through increasing the activation of HDAC, which is subsequently recruited by corticosteroids to suppress inflammatory genes (Cosio et al. 2004; Ito et al. 2002). Its cardiac and central-nervous-system side effects, which occur at doses similar to those required to generate a therapeutic effect, have led to a marked reduction in its use (Holgate and Polosa 2008). More specific immunomodulators that selectively inhibit Th2 lymphocytes have been sought for the treatment of asthma. Suplatast tosilate administration to patients with bronchial asthma inhibited Th2 cells and Th2-type cytokine release and led to the polarization of the circulating Th1/Th2 balance towards the Th1 subset. Japan is the only country in the world where the drug is clinically prescribed (Sano and Yamada 2007; Tanaka et al. 2007).
IMMUNOTHERAPY The sentinel role of IgE in increasing allergen uptake by DCs and activating mast cells and basophils for mediators led to the development of anti-IgE monoclonal antibodies. The humanized IgG1 antibody omalizumab, specific to the CH3 domain of IgE, is now available. It blocks IgE binding to FcεRI and FcεRII. Clinical trials have shown that omalizumab administered subcutaneously 2–4 times per week (depending the total level of IgE in the patient’s plasma and the patient’s body weight) improves symptom control and allows patients to be treated with lower doses of inhaled corticosteroids. Omalizumab is also effective in the
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Table 1. Some of the compounds for asthma treatment (modified from Adcock et al. 2008) Target
Function
Drug
β2 adrenergic receptor
Ultra-long bronchodilation
Indacaterol, carmoterol
Glucocorticoid receptor
Anti-inflammatory
CRTh2 inhibitors
Th2 cell recruitment and activation
CCL11
Blocks eosinophil recruitment/activation
CCR3
Blocks eosinophil recruitment/activation
IgE
Blocks IgE binding to FcεRI and FcεRII
Omalizumab
IL-5
Blocks eosinophil recruitment/activation
Mepolizumab
IL-10
Endogenous anti-inflammatory agent
IL-13
Key driver of asthmatic inflammation
JNK
Anti-inflammatory
CD23
Reduces IgE
Lumiliximab
Sphingosine-1 phosphate receptor
Prevents DC activity
FTY720
VDR
Increased IL-10 expression in Treg cells
Vitamin D3
treatment of allergic rhinoconjunctivitis, but therapy has to begin long before the pollen season (Holgate et al. 2005; Holgate and Polosa 2008). Omalizumab effectively neutralizes IgE. It is unlikely, however, to affect the long-lived plasma cells that express little of the membrane form of IgE. While the tendency of allergic individuals to mount Th2-cell responses in their target organs persists, there is also the likelihood of relapse if the treatment with the antibody is withdrawn (Gould and Sutton 2008). Low-affinity IgE receptor (CD23) plays an important role in the regulation of IgE synthesis. Inhibition of its activity could thus be a promising candidate therapeutic option for the future treatment of allergic diseases. In fact, a CD23-specific antibody, known as lumiliximab, was developed. It has already shown its efficacy in a phase I clinical trial for the treatment of asthma (Rosenwasser and Meng 2005). Because of the principal role of Th2 cytokines in orchestrating allergic inflammation, they and their receptors can be natural therapeutic targets as well. IL-4 and IL-13 are crucially involved in the development of allergic responses. Their biological activities start to be realized when bound to their particular receptors. IL-4 receptor (IL-4R) is a heterodimer consisting of its own α chain (IL-4Rα) and the common γ-chain (γc). IL-13 receptor is also a heterodimer; it shares the IL-4Rα chain in combination with its specific chain (IL-13α1). IL-4 signals through both types of receptors, i.e. IL-4Rα–γc and IL-4Rα–IL-13Rα1. A soluble recombinant IL-4Rα protein (altrakincept), a humanized IL-4-specific monoclonal antibody (pascolizumab), and an IL-4 variant (pitrakinra) that targets allergic Th2 inflammation by potently inhibiting the binding of IL-4 and IL-13 to IL-4 α receptor complexes have been developed. Unfortunately, all of them failed or had only a weak effect in the treatment of asthma, thereby raising doubts over the usefulness of IL-4
Ramatroban
Pitrakinra
blockade in treating already established allergic disease (Borish et al. 1999; Hart et al. 2002; Hart et al. 2001; Wenzel et al. 2007). A humanized anti-IL-5 monoclonal antibody (mepolizumab) has also been developed. Similarly to anti-IL-4 monoclonal antibody, it had no effect on any measures of asthma outcome in the treatment of patients with severe persistent asthma (Holgate and Polosa 2008; Leckie et al. 2000). Based on the finding that levels of TNF-α are increased in the airways and in blood mononuclear cells in severe asthma, a soluble p75 TNF-α receptor fusion protein, etanercept, has been efficiently used for treatment (Howarth et al. 2005; Russo and Polosa 2005). Large multi-center trials with etanercept and TNF-α-specific monoclonal antibodies are now in progress (Holgate and Polosa 2008). Prostaglandin D2, the ligand for the G protein-coupled receptors DP1 and CRTH2, has been implicated in the pathogenesis of the allergic response. It was shown that the administration of a highly potent and specific antagonist of CRTH2 to a mouse model of airway inflammation reduced antigen-specific IgE, IgG1, and IgG2a antibody levels as well as decreased mucus deposition and leukocyte infiltration in the large airways (Lukacs et al. 2008). These findings suggest a possible new way in the treatment of asthma patients. Similarly, there are reports on anti-IL-9 monoclonal antibody treatment (Gaga et al. 2007). IL-9 was proved to be produced by a novel CD4+-subpopulation of T cells (Veldhoen et al. 2008) and IL-9 plays a significant role in driving allergic inflammation (see above). A summary of some compounds used or in development for the treatment of asthma is given in Table 1. Subcutaneous allergen-specific immunotherapy (SCIT) involves the regular subcutaneous injection of allergen extracts or recombinant allergens using incremental regimens. After repeated exposure to allergen(s), SCIT decreases the recruitment of mast cells, basophils,
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and eosinophils in the skin, nose, eye, and bronchial mucosa. SCIT produces an increase in the level of allergen-specific IgA and IgG4 antibodies and a decrease in the level of allergen-specific IgE antibodies. The induction of tolerance takes from several days to several months. However, once tolerance is induced it can last for several years without further treatment (Durham et al. 1999). The limiting factor in SCIT is anaphylactic side effects, which vary in incidence from 0.1–5% of individuals, depending on severity (Williams et al. 2004). Improved efficacy with decreased side effects is the aim of new approaches to SCIT, including chemically modified allergens (allergoids) (Lund et al. 2007). Attaching CpG oligonucleotide motifs to purified allergens also seems to be a particularly promising approach to SCIT by increasing the efficacy and decreasing the side effects, as recently reported for the novel ragweed-allergen conjugate (Creticos et al. 2004). SCIT has been recommended for the treatment of allergic rhinitis, venom hypersensitivity, and mild asthma with only a single or a few allergens involved (Barnes 2008a; Holgate and Polosa 2008). A Cochrane review [www.ginasthma.org., 2008] that examined 75 randomized controlled trials of specific immunotherapy compared with placebo confirmed the efficiency of this therapy in asthma in reducing symptom scores and medication requirements and improving allergen-specific and nonspecific airway hyperresponsiveness. However, in view of the relatively modest effect of allergen-specific immunotherapy compared with other treatment options, these benefits must be weighed against the risk of adverse effects (anaphylaxis induction). Sublingual immunotherapy (SLIT) is the administration of allergens to the oral mucosa. Although much higher doses of allergen are required than those used for SCIT, side effects are rare and mild, which makes this therapy very suitable especially for children. Several clinical trials show that SLIT is effective in the treatment of allergic rhinitis caused by grass, olive, ragweed, and birch pollens as well as rhinitis associated with house dust mite and cat dander allergies (Holgate and Polosa 2008; Pajno 2007). SCIT and SLIT also decrease the development of sensitization to new allergens and decrease the risk of new asthma in both adults and children with rhinitis. Several studies have indicated that allergic rhinitis often precedes asthma and is therefore an important risk factor for the development of asthma (Holgate and Polosa 2008). We can say that both SCIT and SLIT play important roles in the therapy of allergic rhinitis and asthma, but they have to be a part of a complex approach to the patient, including anti-inflammatory and other symptomatic medications.
now clear that the disease also involves local epithelial, mesenchymal, and vascular events that are involved in directing allergic reactions to the lung which eventually result in remodeling of the bronchial wall. Understanding of the immunopathogenesis of the disease resulted in the introduction of new therapeutic approaches, although corticosteroids are still in the mainstay of asthma treatment. However, we still treat the symptoms and do not cure the disease; to achieve this goal, more understanding of genetics, environmental factors, and immunopathogenesis are needed. Acknowledgment: This work was supported by the Grant Agency of Ministry of Education of the Slovak Republic, KEGA 3/7140/09.
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