Humana Press Humana Press Human Airway Inflammation Edited by Duncan F. Rogers Louise E. Donnelly Sampling Techniques and Analytical Protocols Human Airway Inflammation Edited by Duncan F. Rogers Louise E. Donnelly Sampling Techniques and Analytical Protocols M E T H O D S I N M O L E C U L A R M E D I C I N E TM Airway Inflammation and Remodeling in Asthma 1 1 Airway Inflammation and Remodeling in Asthma Current Concepts Stephen T. Holgate 1. Introduction Asthma is a heterogeneous disorder of the airways with intermittent airflow obstruction frequently accompanied by increased responsiveness of the bron- chi to a wide variety of exogenous and endogenous stimuli. A number of dif- ferent types of asthma have been described, based largely on the clinical manifestations of, or factors precipitating, the airflow obstruction. As the dis- ease becomes more severe and chronic, it is suggested that the airflow obstruc- tion may progressively lose some of its reversibility and, in this respect, resemble some aspects of chronic obstructive pulmonary disease (COPD). However, in a small proportion of patients with life-threatening asthma, the airways are highly labile on account of greatly enhanced bronchial hyperresponsiveness. At the other end of the disease severity spectrum there are many patients with mild intermittent asthma whose disease only manifests when exposed to particular sensitizing allergens to which they are sensitive (e.g. outdoor allergens during the pollen season). Over the last 50 yr, the prevalence of asthma and allied allergic disorders have progressively increased on a worldwide scale, in both developed and developing countries. In addition to an increase in asthma-related symptoms, there is evidence of increased medication usage and a rise in hospital admis- sions for asthma. The reasons for these rising trends may be multiple. Sugges- tions include: 1 From: Methods in Molecular Medicine, vol. 56: Human Airway Inflammation: Sampling Techniques and Analytical Protocols Edited by: D. F. Rogers and L. E. Donnelly © Humana Press Inc., Totowa, NJ 2 Holgate 1. Increased allergen exposure (especially within the domestic setting). 2. Reduced exposure to childhood infections. 3. Changes to the diet (e.g., reduced antioxidant and δ 13 polyunsaturated fatty acids). 4. Alterations to the lung or gastrointestinal flora possibly linked to increased anti- biotic prescribing in infancy. Whatever the underlying causes for these rising trends, asthma has now become a public health issue. Therefore, it is of the utmost importance that a clear understanding is obtained of underlying cell and molecular mecha- nisms in order that appropriate biomarkers are identified that can be used to detect the disease earlier and follow its outcome. In childhood and early adulthood, asthma occurs in association with atopy, which is characterized by elevated circulating allergen-specific IgE and positive skin prick test responses to allergen extracts. However, asthma in adults tends to lose its close associa- tion with atopy, particularly in those patients with late onset and chronic dis- ease. Recent estimates suggest that approx 50% of asthma can be linked to immunological mechanisms associated with atopy. Compelling epidemiologi- cal evidence indicates that atopy alone (i.e., the genetic susceptibility to gener- ate allergen-specific IgE) is insufficient for the development of asthma and, what is also required is a susceptibility of the airways to express and respond to localized inflammatory responses. With the recent description of a subtype of asthma manifesting as intermittent cough (cough-variant asthma) in which there is airway inflammation in the absence of bronchial hyperresponsiveness, it would seem that inflammation alone is also insufficient to produce the vari- able airflow obstruction and accompanying symptoms of chronic asthma. It would appear that some alteration to the airway structure (airway wall remod- eling) upon which the inflammatory response is acting is also required. 2. The Epidemiology of Asthma and the Role of IgE Although atopy is the single strongest risk factor for the development of asthma increasing the risk up to 20-fold, only about one-fifth of atopic sub- jects progress to develop chronic asthma requiring regular therapy. Both in childhood and in adults, epidemiological associations have been shown between asthma and IgE, whether assessed as total serum IgE or as allergen- specific IgE. In many parts of the world it is exposure to indoor allergens that appears to drive the expression of atopy linked to asthma and specifi- cally exposure to dust mite, cat, and fungal antigens. Domestic exposure in early life to the major dust mite allergen der P 1 levels of >2 µg/g of house dust has been shown to significantly increase the risk of initial aller- gen sensitization and the development of asthma. Exposure to levels in excess of 10 µg/g of dust increases the risk of acute exacerbations of preexisting dis- ease. Exposure to Alternaria allergens has been linked to acute life-threaten- Airway Inflammation and Remodeling in Asthma 3 ing attacks of asthma, whereas other allergens including animal dander, insect dust, grass pollen, and molds have also been linked to asthma exacerbations. Epidemics of asthma, as occurred in Barcelona or following thunderstorms in the United Kingdom, have also been linked to exposure to high concentrations of allergens. These incidents have been attributed, respectively, to the unload- ing of soy bean in Barcelona harbor and the release of pollen fragments into the air following osmotic lysis of pollen grains at the peak of the pollen season. 3. The Pathological Features of Asthma Asthma is classically an inflammatory disorder of the airways with infiltra- tion of the submucosa and adventitia of both the large and small airways with activated mast cells, tissue macrophages, eosinophils, and, in certain cases, neutrophils. In acute severe asthma that may be provoked by respiratory virus infections, the airways become infiltrated with neutrophils in addition to eosi- nophils, an observation that has also been described in patients dying suddenly of their disease. A second important characteristic of asthma is damage to the ciliated stratified epithelium with a deposition of interstitial collagens (types I, III, and V) as well as laminin and tenascin C beneath the true epithelial base- ment membrane, which has been linked to the proliferation of subepithelial myofibroblasts. The epithelium in asthma also undergoes metaplasia with the acquisition of a repair phenotype and an increase in the number of goblet cells, especially in chronic disease. A third pathological characteristic of chronic asthma is hyperplasia of the formed elements of the airway, including microvessels, afferent neurons, and smooth muscle as well as deposition of matrix proteins and proteoglycans (versican, fibromodulin, biglycan, and decorin) in the submucosa and outside the airway smooth muscle. Taken together, the inflammatory response is superimposed on a remodeled airway, and it is this that gives rise to clinical heterogeneity so characteristic of asthma. Although once considered as purely a disease of airway smooth muscle, asthma is now known to be a chronic inflammatory disorder of the airways, orchestrated by CD4 + lymphocytes. There is evidence to support the view that the asthmatic airway inflammation is driven by the persistence of chroni- cally activated T cells of a memory phenotype (CD45RO + ) with a propor- tion of these being directed to allergenic, occupational, or viral antigens. This is supported by a large number of studies using BAL and bronchial mucosal biopsies from subjects with asthma. These studies revealed increased transcription and product release of a discrete set of cytokines encoded on the long arm of chromosome 5q 31-33 , which includes interleukin (IL)-3, IL-4, IL-5, IL-9, IL-13, and granulocyte macrophage colony stimulating factor (GM-CSF). In more severe asthma and some cases of occupational asthma, CD8 + as well as CD4 + T cells are the source of these cytokines. 4 Holgate Although many of the cytokines associated with the Th-2 phenotype have pleiotropic functions, there are specific aspects of each one that are worth high- lighting when linking their function to airway pathology in asthma. In most types the predominant inflammatory cell infiltrating the airway wall is the eosi- nophil that is derived from CD34 + precursor cells, which are present in the bone marrow, recruited from the circulation, and located in the airway wall itself. Under the influence of IL-3, IL-5, and GM-CSF, eosinophils acquire a mature phenotype with the capacity to secrete a range of preformed and newly gener- ated mediators. Interleukin-4 and IL–13 are intimately involved in the isotype switching of B cells from IgM to IgE and also in the upregulation of a specific adhesion molecule, vascular cell adhesion molecule-1 (VCAM-1), involved in the recruitment of basophils, eosinophils, and Th2-like T cells. Following allergen exposure, it is the interaction of the inflammatory cell integrin VLA-4 with VCAM-1 that plays a key role in the recruitment of eosinophils, baso- phils, and Th-2 cells from the circulation into the airways. An additional prop- erty of IL-4 (which is not shared by IL-13) is the ability of this cytokine to support the development of Th-2 T cells and enhance their survival. Two IL-4 and two IL-13 receptors have been described on a variety of cell types with subtle differences in their ability to initiate intracellular activation mechanisms. These involve the transcription factor signal transducer and activator of tran- scription (STAT)-6 and insulin receptor substrate (IRS)-1/2 that are involved in IL-4/IL-13 induced gene transcription and cell proliferation, respectively. Bronchial epithelial cells and fibroblasts also express IL-4 and IL-13 receptors and are highly responsive to these cytokines in vitro. In vivo overexpression of IL-13 into the bronchial epithelium of transgenic mice not only leads to increased IgE production but also goblet cell metaplasia, subepithelial fibrosis, and bronchial smooth muscle hyperplasia, linked to the acquisition of bron- chial hyperresponsiveness. A Th-2 cytokine that is gaining importance in asthma is GM-CSF, which serves as a growth factor for eosinophils, basophils, macrophages, and dendritic cells and is also a key cytokine for rescuing eosinophils from pro- grammed cell death (apoptosis). 4. An Important Role for Chemokines in Asthma Apart from Th-2 cytokines, a second group of low-molecular-weight pro- teins are required for the recruitment of inflammatory cells into the asthmatic airway — the chemokines. At the transcriptional and protein levels, both C-C and C-X-C chemokines are generated by asthmatic airways but, of particular relevance, are the C-X-C chemokines regulated on activation normal T cell expressed and secreted (RANTES), eotaxin, and monocyte chemoattractant protein (MCP)-1, -3, and -4. These five chemokines interact with the CCR-3 Airway Inflammation and Remodeling in Asthma 5 receptor that is expressed in high numbers on eosinophils, basophils, and a subset of Th-2 cells. Besides promoting local migration of leukocytes within the airway wall, the same chemokines are also involved in the release of eosi- nophils and their precursors from the bone marrow when appropriate signals are received from the lung via the circulation. Although both immune and inflammatory cells have the capacity to release CCR-3 ligands, the most abundant sources of these chemoattractants are the formed airway elements: the bronchial epithelium, microvascular endothelium, and myofibroblast/fibroblast. There is also in vitro evidence to suggest that proliferation of airway smooth muscle can generate chemokines as well as a number of other cytokines, including those encoded in the IL-4 gene cluster. 5. Antigen Processing and Presentation: The Role of Dendritic Cells Activation of the mucosal immune response involving CD4 + T cells is a cen- tral feature of chronic asthma. In order for mucosal T cells to either proliferate and/or generate appropriate cytokines, they require activation usually via the T-cell receptor, CD3. Uptake of antigen by dendritic cells, in allergic disease, is enhanced by the ability of these cells to express both high and low affinity IgE Fc receptors that increase the efficiency of allergen capture by 50–1000-fold. On internalization, a peptide sequence is selected and presented in the groove of major histocompatibility complex (MHC) class II to the T-cell receptor (signal 1). However, in order for T cells to respond to this antigen-specific stimulus, engagement of a second signal is required involving both the costimulatory mol- ecules B7.1 (CD80) or B7.2 (CD86) that engage the homodimeric molecule CD28 on the T-cell (signal 2). In contrast to peripheral blood mononuclear cells that preferentially utilize CD86 for Th-2 cytokine production, CD28 on T cells in the bronchial mucosa of asthmatics engage either CD80 or CD86 in augment- ing local Th-2 cytokine production. In the presence of IL-4, a monomeric adhe- sion molecule CTLA-4 is induced on T cells that has an increased affinity for both CD80 or CD86 and is able to “steal” the CD28 signal from the B7 ligands, thereby rendering the T-cell anergic or initiate its programmed cell death. Dendritic cells are characterized by their cell surface expression of CD1, high density of MHC class II, and costimulatory molecules. They form a net- work within the bronchial epithelium and submucosa. On chronic exposure to antigen, the number of dendritic cells increases, thereby enhancing mucosal responsiveness to sensitizing antigens. These cells are seen to be obligatory for the development of primary allergen-specific airway responses, although, once sensitization has occurred, there are other cells in the airways including B cells and possibly epithelial cells, that may also provide an antigen-processing and -presenting function. 6 Holgate There are strong genetic determinants for the development of Th-2 polariza- tion within the lower airways. Not only do antigen-presenting cells provide T cells with antigen and costimulatory signals, but also soluble signals that polar- ize their subsequent differentiation (signal 3). The Th-2 biased responses observed in atopic diseases appear to be associated with a decreased IL-12/pros- taglandin (PG)-E 2 ratio and, as a consequence, downregulation of Th-2 cytokine production. The presence of interferon-γ (IFN-γ) enhances the ability of imma- ture dendritic cells to produce IL-12, which in turn creates the environment for preferential Th-1 T cell maturation. In contrast, PGE 2 primes for a reduced IL-12, producing ability and consequently, biasing T-cell development in favor of a Th- 2 phenotype. Thus, it is suggested that antigens provoke either a Th-1 or Th-2 response by inducing the production of a pattern of inflammatory dendritic cell mediators with the capacity to direct at the local site of exposure. This concept may be of particular relevance to the early life origins of allergic disease because, in children destined to develop atopic disorders, their cord blood mononuclear cells have an impaired capacity to respond to IL-12 and, as a consequence, to generate IFN-γ efficiently. Because IFN-γ provides such a strong negative signal to Th-2 development, its impaired production may predispose the infant to per- sistence into postnatal life of Th-2 responsiveness that in normal infants shuts down efficiently prior to birth. It has been shown that the impaired production of T-cell IFN-γ production in atopic children persists into late childhood providing further evidence that reduction in this inhibitory pathway rather than enhance- ment of the excitatory is responsible for the persistence of the Th-2 phenotype linked to the allergic phenotype. 6. The Role of IgE The identification of the passive sensitizing agent “reagin” as immunoglobu- lin E has provided the immunological basis for type I hypersensitivity disorders. Immunoglobulin-E directed to specific allergens is generated by B cells and plasma cells through an interaction with antigen-specific T cells in the presence of IL-4 or its homolog (IL-13) together with engagement of the costimulatory molecules CD40 on B cells and CD40 ligand on T cells. Immunoglobulin E binds to both high-affinity Fc receptors (α 1 ,β 1 ,γ 2 : FcεR1) expressed on mast cells, basophils, dendritic cells, and eosinophils and also to monomeric low-affinity receptors (FcεR2 or CD23) expressed on a wide variety of cell types including epithelial cells, B cells, monocytes, T cells, neutrophils, and eosinophils. Although the role of IgE in the manifestation of many allergic diseases such as anaphylaxis and rhinoconjunctivitis is without dispute, there is considerable debate over the role of this signaling molecule and its receptors in asthma. The availability of a fully humanized blocking monoclonal antibody against IgE (E25) has provided considerable insight into the role of IgE-mediated mechanisms in allergic asthma. Airway Inflammation and Remodeling in Asthma 7 This humanized monoclonal antibody was directed to that part of IgE that, under normal circumstances, binds with high affinity to the α chain of the tetrameric FcεR1 receptor. Thus, following a single injection of Mab E-25, there occurs a >90% decrease in circulating levels of IgE with a composite increase in IgE/E25 complexes. These small complexes are rapidly cleared by the reticuloendothe- lial system and, so far, have not been associated with any adverse immune com- plex deposition effects. Over periods of 9–12 wk the regular administration of Mab E25 produced profound attenuation of both the early and late phase bron- choconstrictor responses following allergen provocation of the airways of patients with atopic asthma, as well as causing a reduction in allergen-induced acquired bronchial hyperresponsiveness (BHR). A clinical trial involving over 300 patients in whom E25 was administered over a period of 12 wk has demon- strated improvement in all parameters of asthma, including the requirement of inhaled and oral corticosteroids. Not all patients responded equally to this treat- ment, although there appear to be no particular features that identify “respond- ers” from “nonresponders.” Although IgE has been classically associated with asthma of the atopic extrinsic type, in patients with intrinsic nonallergic asthma Th-2 development in the bronchial mucosa is also accompanied by an increase in the number of cells bearing the FcεR1 receptor. This indicates that local IgE production may contribute to this form of the disease. The putative role of local airway IgE synthesis is further supported by the increased expression of ε germ line tran- scripts (Iε) and mRNA for ε heavy chain of IgE in bronchial biopsies from both atopic and nonatopic asthmatics. However, in the latter case the initiating anti- gen or antigens have yet to be identified. It has been suggested that the inflam- matory response in nonatopic asthma is preferentially promoted by the activation of IgE receptors on monocytes/macrophages rather than mast cells and involves putative “autoimmune” processes. 7. The Role of the Mast Cell in Asthma Immunoglobulin-E-dependent activation of mast cells provides the basis for the early asthmatic response. Crosslinkage of FcεR1 on the surface of sensi- tized mast cells by allergen results in the noncytotoxic secretion of both pre- formed and newly generated mediators. In addition to preformed histamine and heparin, the mast cell secretory granule also contains a range of enzymes, including exoglycosidases, endoglycosidases, and serum proteases. The mucosal type mast cell (MC T ) that predominates in the asthmatic airway con- tains the unique four-chained neutral protease tryptase that is stabilized by heparin. Tryptase has the capacity to cleave a number of soluble substrates, but in asthma its main function may well be to activate protease-activated receptors (specifically PAR-2), through cleavage of a small peptide from the tethered 8 Holgate ligand. This enables the receptor to stimulate cell proliferation and cytokine production. PAR-2 receptors are found on bronchial epithelial, endothelial, neural ganglia, smooth muscle, and fibroblast cells and their function may be to initiate and maintain airway wall remodeling. Activation of PAR-2 recep- tors in the epithelium and endothelium leads to the release of chemokines such as RANTES and IL-8, which may provide one mechanism for chemokine release when sensitized airways are exposed to allergen. In addition to releasing granule-associated preformed mediators, activated mast cells have the capacity to generate an array of newly generated products including the cysteinyl leukotriene LTC 4 and PGD 2 . The combination of LTC 4 , LTD 4 , LTE 4 (together comprising slow reacting substance of anaphylaxis; SRS-A), histamine, and PGD 2 accounts for the majority of the early asthmatic response following allergen challenge. Thus, inhibitors of receptor activation or mediator synthesis, either by themselves or, more effectively, in combina- tion, or mast cell stabilizers, such as sodium cromoglycate and nedocromil sodium, attenuate the early asthmatic response. The same mediators are also implicated in the pathogenesis of exercise-induced asthma. The most popular prevailing hypothesis to explain exercise-induced asthma is that increased ven- tilation impacts on a damaged epithelium resulting in water loss from the air- way lining fluid that is inadequately replaced and, as a consequence, causes activation of primed mast cells by a hyperosmolar environment. Bronchoconstriction provoked by isocapnic ventilation, cold dry air, and hypertonic sodium chloride (or mannitol) aerosols is produced by a similar mast cell-dependent mechanism. Similarly, in asthma, adenosine generated by mast cells (and also by other cells) interacts with adenosine A 2B receptors on primed mast cells leading to autacoid release. H 1 -antihistamines and mast cell suppressor drugs (e.g., sodium cromoglycate and nedocromil sodium) can also inhibit this early response. A characteristic feature of asthma associated with known environmental sen- sitizing agents is the occurrence of delayed bronchoconstriction 4–24 h fol- lowing inhalation exposure, which is accompanied by a progressive increase in bronchial hyperresponsiveness that may last up to 3 wk following challenge. This late asthmatic response is, in part, mast cell dependent because it is also inhibited by sodium cromoglycate and nedocromil sodium and also by nonanaphylactogenic antihuman IgE. The most likely explanation for the onset of a late asthmatic response is the release of cytokines from activated mast cells, specifically TNFα, IL-4, IL-5, GM-CSF, and chemokines active at the CCR3 receptor. Human mast cells store small quantities of cytokines within their secretory granules that can be released rapidly. In addition, activation of their IgE receptors in the presence of stem cell factor (a mast cell growth fac- tor) leads to increased cytokine transcription with subsequent product release Airway Inflammation and Remodeling in Asthma 9 that may persist for up to 48–72 h. Mast cell-derived histamine and leukotrienes express the preformed adhesion molecule P selectin on endothelial cells result- ing in leukocyte “rolling.” TNFα upregulates the expression of the adhesion molecules E-selectin, intercellular adhesion molecule-1 (ICAM-1), and VCAM-1, the latter also requiring either IL-4 or IL-13 for stabilization of expression. By interacting with their complementary ligands on neutrophils, eosinophils, basophils, T cells, and monocytes, these adhesion molecules enable leukocytes to be recruited selectively into the airway wall. There is strong evidence that cytokine release from T cells also contributes to the latter period of the late phase bronchoconstrictor response and the accompanying increase in BHR. Antigen specific T cells have the capacity to be selectively recruited into exposed airways, possibly involving novel chemokine and epithelial homing receptors. 8. The Eosinophil The presence of eosinophils in the walls and lumen of the conducting air- ways is a characteristic feature of chronic asthma and has placed this cell at the center of the mediator cascade in all types of asthma irrespective of etiology. The extent of airway eosinophilia is closely linked to epithelial damage and disease severity, as reflected by eosinophil counts and the presence of granule proteins in lavage fluid, mucosal tissue, and induced sputum. In chronic asthma increased eosinophil survival by locally produced GM-CSF, IL-3, and IL-5 is at least as important in maintaining airway eosinophilia as is the recruitment of new cells from the circulation. More recently, the identification of CD3 + leu- kocyte precursors in the airway wall bearing receptors for the same eosinophilopoietic cytokines suggests that part of the tissue eosinophilia may be locally generated. The eosinophil granules contain a number of arginine rich proteins (major basic protein [MBP]; eosinophil cationic protein [ECP]; eosinophil, derived neurotoxin [EDN]; and eosinophil peroxidase [EPO]). At high concentrations these proteins are cytotoxic to the bronchial epithelium whereas when present in smaller amounts they activate epithelial stress signaling pathways and have the capacity to interfere with muscarinic neurotransmission. It has been sug- gested that MBP and possibly ECP are responsible for the epithelial disruption in asthma. However, there is also evidence that detachment of columnar cells from basal cells occurs through weakening of cell adhesion complexes, possi- bly mediated by an altered epithelial phenotype, proteolytic attack, or increased apoptosis. Eosinophils and neutrophils are rich sources of the metalloproteinase MMP-9 whose inhibition by the endogenous tissue inhibitor TIMP-1 has been shown to be impaired in chronic asthma. MMP-9 is also induced in epithelial cells when they are engaged in a repair response. [...]... assessment with the aid of a computerized image system From: Methods in Molecular Medicine, vol 56: Human Airway Inflammation: Sampling Techniques and Analytical Protocols Edited by: D F Rogers and L E Donnelly © Humana Press Inc., Totowa, NJ 19 20 Saetta and Turato Table 1 Advantages and Disadvantages of the Different Techniques for Processing Bronchial Biopsies for Light Microscopic Analysisa Procedure Advantages... and immediately prepared for microscopic analysis There are various methods of processing biopsy samples: 1 Snap-freezing without prior fixation (3) Biopsy Techniques 23 2 Paraformaldehyde fixation and freezing (13) 3 Formalin fixation and paraffin-embedding (14) 4 Fixation and GMA-embedding (15,16) Bronchial biopsies are potentially hazardous human material, and the same precautions as used in handling... 56: Human Airway Inflammation: Sampling Techniques and Analytical Protocols Edited by: D F Rogers and L E Donnelly © Humana Press Inc., Totowa, NJ 31 32 Ward and Walters BAL is useful diagnostically in assessment of lower respiratory infections and has been particularly useful in immunocompromised patients (10) The technique is also used clinically in oncology and especially in the diagnosis of peripheral... N., et al (1993) Eosinophilic and neutrophilic inflammation in asthma , chronic bronchitis and chronic obstructive pulmonary disease J Allergy Clin Immunol 92, 537–548 6 Bradding, P., Roberts, J A., Britten, K M., Montefort, S., Djukanovic, R., Mueller, R., et al (1994) Interleukin-4,-5, and -6 and tumor necrosis factor-a in normal and asthmatic airways: evidence for the human mast cell as a source of... through the era of airway inflammation and now back to remodeling T-cell polarization toward a Th-2 phenotype and activation of the bronchial epithelial–mesenchymal trophic unit are essential prerequisites for the development of asthma, leading to sustained airway inflammation and airway wall remodeling These processes are likely to be under strong genetic influences Although a number of candidate genes... risk of asthma and atopy be reduced by a vaccine that induces a strong T-helper Type 1 response? Am J Respir Cell Mol Biol 2000; 22:139–142 Warner JO, Pohunek P, Marguet C, Roche WR, Clough JB Issues in understanding childhood asthma J Allergy Clin Immunol 2000; 105 (pt.2): S473–S476 Biopsy Techniques 19 2 Biopsy Techniques Optimization for Collection and Preservation Marina Saetta and Graziella Turato... cells promoting airway growth and branching Key cytokines involved in this signaling include members of the epidermal growth factor family (EGF itself, amphiregulin, HB-EGF, β cellulin, and epiregulin), b-FGF, endothelin-1, PDGF, and IGF-1 In the reverse direction, myofibroblasts and fibroblasts are important sources of keratinocyte growth factor (KGF), acid fibroblast growth factor (a-FGF), and PDGF Both... as an important formed element of the airway, may not be alone in orchestrating inflammatory and repair responses characteristic of Airway Inflammation and Remodeling in Asthma 13 asthma A unique and highly characteristic feature of asthma is the deposition of interstitial collagens secreted by myofibroblasts beneath the basement membrane The bronchial epithelium and subepithelial mesenchymal cells are... tissue Airway Inflammation and Remodeling in Asthma 15 14 Concluding Comments The challenge for the future will be to bring together the various methodological approaches for studying airway inflammation and remodeling in asthma toward a common objective of identifying novel gene products which underpin asthma susceptibility and disease progression engaging both altered mucosal immune responses, inflammation,. .. Y M., Sloan, S I., Rex, M D., and Martin, R J (1997) Bronchoscopic evaluation of severe asthma Am J Respir Crit Care Med 156, 737–743 8 Kraft, M., Djukanovich, R., Torvik, J., et al (1995) Evaluation of airway inflammation by endobronchial and transbronchial biopsy in nocturnal and non-nocturnal asthma Chest 107, 162S 9 Laitinen, L.A., Laitinen, A., and Haahtela, T 1993 Airway mucosal inflammation even . Humana Press Humana Press Human Airway Inflammation Edited by Duncan F. Rogers Louise E. Donnelly Sampling Techniques and Analytical Protocols Human Airway Inflammation Edited. Donnelly Sampling Techniques and Analytical Protocols M E T H O D S I N M O L E C U L A R M E D I C I N E TM Airway Inflammation and Remodeling in Asthma 1 1 Airway Inflammation and Remodeling in. 56: Human Airway Inflammation: Sampling Techniques and Analytical Protocols Edited by: D. F. Rogers and L. E. Donnelly © Humana Press Inc., Totowa, NJ 20 Saetta and Turato There are various methods