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Modeling allergic asthma: from in vitro assays to virtual patients
Vol. 1, No. 4 2004
Drug Discovery Today: Disease Models
DRUG DISCOVERY
TODAY
DISEASE
MODELS
Editors-in-Chief Jan Tornell – AstraZeneca, Sweden Denis Noble – University of Oxford, UK
Immunological disorders
Modeling allergic asthma: from in vitro assays to virtual patients Michelle M. Epstein Medical University of Vienna, Department of Dermatology, Experimental Allergy, Lazarettgasse 19, A-1090 Vienna, Austria
Allergic asthma is a complex disease of the respiratory tract characterized by allergen-induced breathlessness that is, orchestrated by the interaction of immune, inflammatory, and resident lung cells. Great strides have been made with in vitro and in vivo experimentation, but disease pathogenesis remains obscure. The combination of cellular, in vivo, and in silico modeling promises the eventual creation of a heterogeneous population of virtual patients to pursue unresolved
Section Editor: Veena Taneja – Mayo Clinic, Rochester, USA The growing epidemic of allergic asthma has led to researchers developing in vitro and in vivo models for understanding the pathogenesis of this chronic inflammatory disease. These models have advanced our knowledge of the immunology and genetic susceptibility of the disease. However, in vitro models exclude microenvironmental influences and in vivo animal models cannot replicate the human disease. The new humanized models have the advantage of delineating the role of human cells and genes, although the effects of the xenogeneic microenvironment cannot be ruled out. Current studies should lead to new insights into the pathogenesis of allergic asthma.
issues in allergic asthma. Pathogenesis Introduction Allergic asthma is a syndrome characterized by shortness of breath, wheezing, coughing, and increased mucus secretions upon exposure to allergen. The type of allergen exposure determines whether symptoms are intermittent (e.g. exposure to cats), seasonal (e.g. ragweed, grass pollens) or chronic (e.g. dust mite) and the clinical course of disease might be mild, severe, and in some cases fatal. Allergic asthma is a major burden on patients, family, and society at large. It is associated with enormous health care costs, millions of lost workdays, school days, and might lead to a chronic, debilitating illness requiring long-term care. Despite a better grasp of the fundamental mechanisms of allergic asthma, a complete understanding of the pathogenesis, genetic susceptibility, and the increase in incidence and prevalence remains elusive. This review presents an overview and comparative analysis of experimental models of allergic asthma with a focus on developments in the past year. E-mail address: (M.M. Epstein) [email protected]. 1740-6757/$ ß 2004 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddmod.2004.11.009
The role of the immune system is to protect the host against foreign pathogens. However, sometimes the immune system reacts against non-infectious, otherwise harmless foreign proteins, i.e. pollen allergens. This type of adaptive (acquired) immune response begins with the uptake of protein allergens by antigen presenting cells (APC), processing of the protein into small peptide fragments in these cells, and then transport as a peptide: MHC class II complex to the cell surface (Fig. 1). The T cell receptor (TCR) on CD4+ T helper (Th) lymphocytes interacts with this complex resulting in T cell activation and differentiation. Subsequently, activated effector CD4+ Th cells ‘help’ B cells produce antibody and produce Th2-type (IL-4, IL-5, IL-13) cytokines that initiate recruitment of eosinophils, B cell production of IgE, and mucus hypersecretion by goblet cells in the airways. The reason why allergic responses are generated in certain individuals is unknown. Moreover, it is also not clear whether all ‘susceptible’ individuals develop allergic immune responses. Normally allergenic proteins do not elicit pathogenic immune responses in non-allergic individuals. There are three possible www.drugdiscoverytoday.com
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Figure 1. Allergen-specific adaptive immune response leading to allergic asthma. Allergen is taken up by antigen presenting (in this case the macrophage) cells, macrophages or DCs in the lungs. It is then processed into peptide fragments that are complexed within the cell with MHC class II and brought to the cell surface. The complex then interacts with the TCR on CD4+ T cells resulting in T cell activation, clonal expansion, alteration of cell surface molecule phenotype, and the production of cytokines. These cytokines then induce the differentiation and recruitment of eosinophils to the lungs and stimulate the production of IgE by B cells. The IgE produced binds allergen and crosslinks the Fce receptors on mast cells causing the release of vasoactive mediators. The combination of eosinophils, products from mast cell degranulation, and Th2 cytokines leads to smooth muscle contraction, mucus hypersecretion, edema, and an inflammatory infiltrate in the lungs. Repeated allergen exposure results in recurrence of disease and eventually might lead to permanent lung changes, termed airway remodeling, and lung destruction.
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explanations including: (1) non-susceptible individuals generate a non-allergic response, (2) they ignore the allergen, that is do not develop any type of response, and (3) they develop immunological tolerance (a process that causes immune unresponsiveness). Following sensitization, subsequent encounters with allergen induce more vigorous and rapid responses, which are initiated by Th2 memory cells (a feature of adaptive immunity) that reside in the lungs and by the release of vasoactive mediators by mast cells upon allergen-IgE crosslinking of cell surface Fce-receptors. The result is reversible airway obstruction caused by edema, increased mucus production, smooth muscle cell contraction, and eventual lung damage.
In vitro models of allergic asthma The adaptive and innate immune systems (T-lymphocytes, Blymphocytes, mast cells, eosinophils, macrophages, dendritic cells) and resident lung cells (respiratory epithelial, smooth muscle, and nerve cells) are all involved in allergic asthma. The relatively easy access to immune cells allows them to be isolated and purified from peripheral and cord blood, and respiratory tract cells can be isolated from sputum, nasal or bronchial washings, and biopsies, and subsequently cultured and evaluated for the expression of cell surface molecules, ability to proliferate, migrate, apoptose, and secrete. Typically, in vitro models utilize cultured single cell suspensions, however, newer models cultivate respiratory tract tissue explants.
Adaptive immunity Adaptive immunity relates to antigen-specific defense mechanisms that are designed to eliminate antigen and are maintained throughout life. The majority of in vitro models of allergic asthma involve cells of the adaptive immune system specifically Th2 cells from blood and the respiratory tract because they play a central role in the initiation and maintenance of immune responses to allergens. In vitro stimulation of T cells with allergen, mitogens, environmental agents, and antibodies directed against the TCR leads to cell proliferation, cytokine/chemokine production, up- or down-regulation of cell surface molecules, and migration [1–3]. Additionally, it is possible to transfect T cells with a specific molecule to determine its effect on cell function. A recent example of this approach involved the transfection of the T-bet transcription factor into isolated Th2 cells [4], which significantly altered cytokine production and migration properties of Th2 cells. To simplify analysis of peripheral T and B cells in vitro, it is possible to use whole blood [5], peripheral blood mononuclear cells (PBMC) [6–8], and cultured cord blood mononuclear cells [9]. The advantage of these approaches is the avoidance of further lymphocyte isolation and purification to determine function and phenotype. One of the latest advances in T cell biology involves the
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differentiation of T cells from embryonic stem cells in vitro [10]. It is tempting to speculate that new models of allergic asthma will evolve from in vitro differentiated stem cells from allergic individuals.
Innate immunity The cells of the innate and inflammatory immune system including lung macrophages eosinophils, basophils, mast cells, dendritic cells, and neutrophils are involved in initiation and perpetuation of allergic inflammation and probably lung tissue destruction. They can be cultured and studied in vitro. For instance, blood eosinophils purified and stimulated in vitro by allergen and cytokines, can be assessed for migration, reactive oxygen species production, survival and viability, and cell surface molecule expression [7,11,12]. In a recent investigation of neutrophil function, specific allergens stimulated degranulation and release of elastase, suggesting a potential role of neutrophils in allergic inflammation [13]. APC (macrophages and dendritic cells) play a crucial role in allergic immune responses. Although APC can be purified, isolated, and generated from CD34+ peripheral blood monocytes, their function can be evaluated indirectly by measuring the IL-12 production by PBMC in vitro. In a recent study, investigators correlated reduced PBMC-IL-12 production in the perinatal period with an increased propensity to develop allergic type immune responses and implied that APC function early in life has an impact on allergic susceptibility in later life [14]. Although most cells studied in vitro tend to be differentiated effector cells, early progenitors migrating from the bone marrow to tissue provide a constant source of effector cells during an allergic inflammatory response. Bone marrow-derived CD34+ stem cells from asthmatics stimulated with allergen [15] and stimulated cord blood CD34+ progenitor cells [16] can differentiate into eosinophils. In vitro analysis of cultured cord blood cells from offspring of allergic and non-allergic parents might predict the development of allergic disease [17]. In vitro evaluation of innate immune cells in the context of adaptive immunity is a promising approach for elucidating disease pathogenesis and target identification.
Resident lung cells Mucus hypersecretion, smooth muscle contractility, hypertrophy and hyperplasia, and secreted chemokines, cytokines and growth factors from epithelial cells play crucial roles in airway obstruction and allergic inflammation. Epithelial and muscle cells are harvested and cultured from biopsy specimens. The response of primary cultured airway smooth muscle cells can be studied in response to stimuli [18]. Additionally, bronchial epithelial cells can be cultured and assessed for chemokine and cytokine production [19]. Moreover, the effects of byproducts of inflammation, i.e. secreted cationic proteins from eosinophils on the epithelium can be studied in vitro [20]. Culturing primary epithelial cells from www.drugdiscoverytoday.com
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guinea pig trachea in the presence of IL-13 results in mature goblet cells and is a promising model to study mucus production [21]. An important advance in the study of bronchial epithelium is the production of human bioengineered bronchial equivalents [23]. Healthy or asthmatic human bronchial epithelial and fibroblastic cells from biopsy specimens cultured on a mesenchymal layer undergo differentiation resulting in ciliated and goblet cells in a pseudostratified epithelium that resembles human bronchi. Insights on the physiological and pathophysiological responses of bronchial epithelium and smooth muscle will enable the further understanding of the complex interaction between allergic response and respiratory tract during allergic asthma.
Tissue explants The advantages of studying human tissue explant models of allergic asthma rather than cell suspensions is that the microenvironment is maintained and it is possible to measure inflammatory cell function [23,24], mucus production [25], and bronchial constriction [26]. Remarkably, allergeninduced cytokine responses of bronchial explants and PBMC from patients with asthma lead to disparate results [23], suggesting that it is necessary to determine which model is more predictive. Another advance in the field of tissue explants involves the use of human precision-cut lung slices [26]. The method utilizes 250 mm slices resected from lungs, cultured with serum from allergic patients and allergen or IgE antibody, in the presence or absence of antagonists, and videomicroscopy to record bronchoconstriction of bronchioles.
In vivo models of allergic asthma Although in vitro assays are useful and have made significant advances in the elucidation of cellular events in allergic asthma, the complex mechanisms underlying sensitization, genetics, and pathophysiology necessitate in vivo studies that cannot be done in humans. Fortunately, animal models of allergic asthma mimic clinical disease (reviewed in [27]). There are numerous models in mice, rats, dogs, cats, monkeys, and sheep, but small rodent models tend to be more widely used, owing to lower research costs and the availability of a large variety of transgenic animals and reagents. Models are generated with many different antigens, in the
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presence or absence of adjuvant, and with differing protocols. They address the clinical course, long-term effects of disease, airway remodeling, immune tolerance, prenatal sensitization, and immunological memory. Furthermore, these models can be used for drug discovery and evaluation of treatment effectiveness during distinct stages of disease (Fig. 2).
Allergen-induced animal models The majority of in vivo models of allergic asthma are induced by immunizing and challenging the respiratory tract with antigen or allergen. Some examples of animal models in use include, Bermuda grass allergen-induced feline asthma [28], ovalbumin (OVA)-induced disease in guinea pigs [29,30], mice [27], and pollen-allergic dogs [31]. The models are characterized by eosinophilic lung inflammation, mucus hypersecretion, airway hyperresponsiveness (AHR), and elevated production of IgE. Although the most commonly used model is in mice, and by and large, reflects clinical disease, there are notable differences, which include a less crucial role of mast cells and IgE, and a transient rather than sustained increase in airway reactivity [27].
Spontaneous disease models An ideal animal model of allergic asthma is one that spontaneously develops disease. Two examples of naturally occurring allergic asthma models are Ascaris suum-allergic sheep [32] and flea-allergic dogs [8]. These models offer the opportunity to study disease initiation, genetic inheritance, and pathogenesis and have certain advantages; the animals can be followed long term because they can undergo repeated bronchoalveolar lavage (BAL), biopsy, AHR procedures, and can be examined using clinical radiological and other scanning techniques. Another spontaneous asthma model was generated recently in mice [33]. Investigators produced T-bet knockout mice that develop AHR, lung inflammation, and features of airway remodeling without allergen exposure. Although these mice provide novel experimental opportunities, it is necessary to determine whether this model represents allergic asthma or non-allergic asthma.
Humanized animal models To avoid the constraints of studying human cells in vitro and animal physiology, which does not exactly replicate the
Figure 2. Treatment intervention in an allergic asthma in vivo model. (a) BALB/c or C57BL/6 mice immunized with OVA (10 mg, without adjuvant) intraperitoneally (i.p.), 3 weeks apart generates a Th2 immune response and OVA-specific IgE. (b) One week later, mice are placed in a plastic box and aerosolized with a 1% solution of OVA with an ultrasonic nebulizer for 1 h twice daily for 2 consecutive days. Within 2 days (peaks on day 31), mice develop eosinophilic lung inflammation, mucus hypersecretion, and AHR. Mice recuperate from acute allergic asthma within 30 days of the last aerosol. (c) At anytime during their lifetime, after having recovered from acute disease, the mice will develop an allergic asthma relapse upon exposure to nebulized OVA. Underlying this immunological memory response is the life-long persistence of lung inflammatory infiltrates containing Th2 memory cells. This is a useful in vivo mouse model for drug discovery and for testing drug effectiveness. Treatment intervention is possible before sensitization (a), before the clinical manifestations of allergic asthma (b), and before disease relapse (c).
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Table 1. Comparison summary table In vitro models
In vivo models
In silico models
Pros
Easy access to cells Reduced complexity Utilization of human tissue Avoids the need for animals Individual cell responses can be measured Respiratory tract tissue explants now available
Complex system that more accurately resembles human disease Multiple variables can be evaluated Many animal models: including spontaneous and humanizedmouse models Able to study disease initiation, pathogenesis, susceptibility and in vivo treatment efficacy Small animals have shorter lifespans which enable studies on long-term disease progression Armamentarium of transgenic animals
Modeling of metabolic pathways, receptor–ligand drug interactions, treatment effectiveness Complex disease modeling is possible Improves speed of drug development Decreases potential drug adverse side effects, predicts response Might improve understanding of physiology Possible to generate virtual patients
Cons
Excludes microenvironment influences and might be misleading
Animal models are not exact replicas of human disease
Explants exclude macroenvironmental influences Long-term effects cannot be observed Bronchial biopsies are not trivial, raise ethical concerns
Transgenic knockout mice generated using different techniques might lead to disparate results Larger animal studies are expensive, less available, longer lifespans
Simulation models reflect what is known; if the mechanism is not entirely clear, the model might be erroneous
Evaluation of cell physiology, function, viability, phenotype, and responses to stimulators and inhibitors
Evaluation of disease initiation, host susceptibility Perform pre-clinical treatment studies Evaluation of chronic disease and lung remodeling
Best use of model
References
[1–25]
Ethical considerations regarding the use of animals in research
[26–36]
clinical situation; several laboratories have established human–mouse chimeric models. PBMC from allergic patients injected into B and T lymphocyte deficient severe combined immunodeficiency (SCID) mice produce the human-SCID (hu-SCID) mouse model. Although most cells belong to the recipient mice, many cells, including lymphocytes and dendritic cells are of human origin. When hu-SCID mice are aerosolized with the offending allergen, they develop allergic asthma characterized by human IgE, pulmonary inflammation, AHR [34,35], and airways and lungs infiltrated with human T lymphocytes [36] and dendritic cells [37]. Although this model offers advantages, it is unclear whether human cells function in mice exactly as they do in humans. In another human–mouse model, investigators utilized a clinically relevant allergen, short ragweed, Ambrosia artemisiifolia, in transgenic mice expressing human MHC class II (DQ8;HLADQA1*0301 and HLA-DQB1*0302). This model offers the opportunity to evaluate human HLA class II influences and the roles of other T cell and APC molecules, such as, CD28/B7 in allergic asthma [38].
In silico models of allergic asthma Computer simulations used to generate virtual ligand–receptor interactions, cells, respiratory tracts, and patients are the newest wave in modeling allergic asthma. 392
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Simulations of cell responsiveness, disease induction, factors involved in generating chronic disease, prediction of drug treatment efficacy, adverse side effects [37,38]
Virtual drug discovery In silico screening to identify novel drugs might become a useful tool. An example of this approach in allergic asthma is a computational screening of ligand–receptor interactions based on 3D structure of the X-ray analysis of a particular binding site. This creates a 3D ‘pharmacophore’ model, which generates a pattern of spatial and chemical constraints of the ligand. This approach was used to produce non-peptidic antagonists of VLA-4, an adhesion molecule involved in cell–cell interactions [32]. The compounds identified from a virtual library were chosen on the basis of an IC50 comparable to the original ligand. The synthesized compounds were then tested in an allergic sheep model of asthma. This is an example of an integrative approach of in silico, pharmacological, and in vivo models.
Virtual organs A variety of computer simulations of lungs have led to the socalled, ‘lung physiome’ or ‘virtual lung’. These simulations provide physiological measurements of healthy and asthmatic lungs, and can be used for testing drug efficacy. One example is the simulation of a 3D human respiratory tract using MRI and SPECT images from lungs and thorax. The virtual respiratory tract enables researchers to evaluate lumen diameter based on bronchoconstriction, inflammation, and
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mucosal thickness and has been used to predict the efficiency of aerosol drug delivery in healthy versus asthmatic subjects [39].
Virtual patients The virtual allergic asthmatic promises to be the next generation of in silico model. One example is the virtual rat lung model developed to determine the effects of environmental pollution on lung function (www.pnl.gov). A second example is a computer-based mathematical model of airway structure and function, inflammation, and immune responses to create virtual asthmatic patients (Entelos1 Asthma PhysioLabTM) [40] by the company, Entelos Inc., Menlos, CA, USA (www.entelos.com). A major aim of virtual patient models is to reduce the costs of getting new drugs to the market. However, there is growing interest for these models to be used to study disease pathogenesis. An example is the physiome project (www.physiome.org), which is dedicated to the generation of databases from existing knowledge to produce physiomes or virtual computer-generated models of humans and other animals to study pathophysiology.
Model comparison There is no ‘best’ model of allergic asthma. Taken together, in vitro, in vivo, and in silico models of allergic asthma are constantly accumulating information to increase the knowledge about initiation and perpetuation of disease, genetic susceptibility, pathogenesis, effectiveness of therapy, and to find better drugs. Individually, the models have strengths and weaknesses that need to be acknowledged (Table 1). Cellular in vitro assays exclude the effect of microenvironment influences and cannot recapitulate disease complexity. By contrast, tissue explant models include microenviroment but exclude macroenvironment influences. Additionally, the discrepancy between PBMC and explant results suggests that old questions need to be revisited. Another concern is that in vitro lung tissue models require bronchial biopsy, which is a safe but not trivial procedure. Moreover, it is rarely clinically indicated for asthmatics and thus, raises ethical considerations. In vivo models are necessary because of the complexity of disease. Furthermore, cell and explant assays cannot completely reproduce the conditions necessary for understanding allergic asthma. The allergen-induced and spontaneous animal models are important, but they are not exact replicas of human disease and thus, are not ideal. Humanized-mouse models combine the advantages of both human cells and in situ microenvironment, but have not excluded xenogeneic influences. Additionally, heavy reliance on in vivo models, especially genetically mutated knockout mice might be unwise. Recently, two laboratories established mice lacking eosinophils to study their role in allergic asthma [41,42]. They used different experimental strategies and generated conflicting results. The reason for these disparate observa-
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tions is not known but is possibly owing to mouse strain differences or related to the approach used to delete the eosinophils. Although these findings have not definitively elucidated a role for eosinophils in allergic asthma, they illustrate that transgenic in vivo models must be interpreted cautiously. In silico models have potential in allergic asthma. Modeling of ligand–receptor interactions offer a valuable approach for drug design and organ modeling has important implications for lung physiology and drug discovery. An exciting possibility is comparative studies of virtual healthy and asthmatic patients. However, a major problem is that the generation of simulated humans is limited by the extent of existing knowledge. The model can only be as accurate as the database.
Conclusions Many of the advances in allergic asthma have been the result of an interrelationship between in vitro and in vivo modeling. Importantly, in vitro observations are confirmed in vivo and vice versa. This establishes a phenomenon that might be followed by the generation of a transgenic knock in or knock out in vivo model. The combination of data from all models might then be used as the basis for clinical and epidemiological studies in humans. The hope is that in silico modeling and the generation of virtual patients will eventually reduce the need for clinical studies. In summary, a complementary approach incorporating experimental observations from in vitro, in vivo, and in silico models is necessary. A daunting task will be the gathering, evaluating, integrating, and ultimately synthesizing all available data.
Acknowledgements I would like to thank B. Mittleman, P. Stuetz and O. Hoffmann for their critical reading of the manuscript.
Outstanding issues
When and how is allergic asthma initiated? Who is susceptible to allergic disease? What factors influence disease susceptibility? What is the effect of repeated disease exacerbations on lung function? What role do environmental factors (other than allergen) play in allergic asthma? Why is incidence and prevalence increasing in the industrialized world?
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3 Senechal, S. et al. (2003) Effect of diesel on chemokines and chemokine receptors involved in helper T cell type 1/type 2 recruitment in patients with asthma. Am. J. Respir. Crit. Care Med. 168, 215–221 4 Lametschwandtner, G. et al. (2004) Sustained T-bet expression confers polarized human TH2 cells with TH1-like cytokine production and migratory capacities. J. Allergy Clin. Immunol. 113, 987–994 5 Grob, M. et al. (2003) Altered intracellular expression of the chemokines MIP-1alpha, MIP-1beta and IL-8 by peripheral blood CD4+ and CD8+ T cells in mild allergic asthma. Allergy 58, 239–245 6 El-Mezayen, R.E. and Matsumoto, T. (2004) In vitro responsiveness to IL18 in combination with IL-12 or IL-2 by PBMC from patients with bronchial asthma and atopic dermatitis. Clin. Immunol. 111, 61–68 7 Frieri, M. et al. (2003) Montelukast inhibits interleukin-5 mRNA expression and cysteinyl leukotriene production in ragweed and mite-stimulated peripheral blood mononuclear cells from patients with asthma. Allergy Asthma Proc. 24, 359–366 8 Tang, L. et al. (2003) Expression and characterization of recombinant canine IL-13 receptor alpha2 protein and its biological activity in vitro. Mol. Immunol. 39, 719–727 9 Nilsson, C. et al. (2004) Low numbers of interleukin-12-producing cord blood mononuclear cells and immunoglobulin E sensitization in early childhood. Clin. Exp. Allergy 34, 373–380 10 Schmitt, T.M. et al. (2004) Induction of T cell development and establishment of T cell competence from embryonic stem cells differentiated in vitro. Nat. Immunol. 5, 410–417 11 Myrtek, D. et al. (2004) Expression of interleukin-13 receptor alpha 1subunit on peripheral blood eosinophils is regulated by cytokines. Immunology 112, 597–604 12 Davoine, F. et al. (2004) Role and modulation of CD16 expression on eosinophils by cytokines and immune complexes. Int. Arch. Allergy Immunol. 134, 165–172 13 Monteseirin, J. et al. (2003) Specific allergens enhance elastase release in stimulated neutrophils from asthmatic patients. Int. Arch. Allergy Immunol. 131, 174–181 14 Prescott, S.L. et al. (2003) Neonatal interleukin-12 capacity is associated with variations in allergen-specific immune responses in the neonatal and postnatal periods. Clin. Exp. Allergy 33, 566–572 15 Sehmi, R. et al. (2003) Allergen-induced fluctuation in CC chemokine receptor 3 expression on bone marrow CD34+ cells from asthmatic subjects: significance for mobilization of haemopoietic progenitor cells in allergic inflammation. Immunology 109, 536–546 16 Lamkhioued, B. et al. (2003) The CCR3 receptor is involved in eosinophil differentiation and is up-regulated by Th2 cytokines in CD34+ progenitor cells. J. Immunol. 170, 537–547 17 Hoffjan, S. et al. (2004) Genetic variation in immunoregulatory pathways and atopic phenotypes in infancy. J. Allergy Clin. Immunol. 113, 511–518 18 Gounni, A.S. et al. (2004) IL-9-mediated induction of eotaxin1/CCL11 in human airway smooth muscle cells. J. Immunol. 173, 2771–2779 19 Komiya, A. et al. (2003) Concerted expression of eotaxin-1, eotaxin-2, and eotaxin-3 in human bronchial epithelial cells. Cell. Immunol. 225, 91–100 20 Shahana, S. et al. (2002) Effects of the cationic protein poly-L-arginine on airway epithelial cells in vitro. Mediators Inflamm. 11, 141–148 21 Kondo, M. et al. (2002) Interleukin-13 induces goblet cell differentiation in primary cell culture from Guinea pig tracheal epithelium. Am. J. Respir. Cell. Mol. Biol. 27, 536–541
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Drug Discovery Today: Disease Models
DRUG DISCOVERY
TODAY
DISEASE
MODELS
Editors-in-Chief Jan Tornell – AstraZeneca, Sweden Denis Noble – University of Oxford, UK
Immunological disorders
Modeling allergic asthma: from in vitro assays to virtual patients Michelle M. Epstein Medical University of Vienna, Department of Dermatology, Experimental Allergy, Lazarettgasse 19, A-1090 Vienna, Austria
Allergic asthma is a complex disease of the respiratory tract characterized by allergen-induced breathlessness that is, orchestrated by the interaction of immune, inflammatory, and resident lung cells. Great strides have been made with in vitro and in vivo experimentation, but disease pathogenesis remains obscure. The combination of cellular, in vivo, and in silico modeling promises the eventual creation of a heterogeneous population of virtual patients to pursue unresolved
Section Editor: Veena Taneja – Mayo Clinic, Rochester, USA The growing epidemic of allergic asthma has led to researchers developing in vitro and in vivo models for understanding the pathogenesis of this chronic inflammatory disease. These models have advanced our knowledge of the immunology and genetic susceptibility of the disease. However, in vitro models exclude microenvironmental influences and in vivo animal models cannot replicate the human disease. The new humanized models have the advantage of delineating the role of human cells and genes, although the effects of the xenogeneic microenvironment cannot be ruled out. Current studies should lead to new insights into the pathogenesis of allergic asthma.
issues in allergic asthma. Pathogenesis Introduction Allergic asthma is a syndrome characterized by shortness of breath, wheezing, coughing, and increased mucus secretions upon exposure to allergen. The type of allergen exposure determines whether symptoms are intermittent (e.g. exposure to cats), seasonal (e.g. ragweed, grass pollens) or chronic (e.g. dust mite) and the clinical course of disease might be mild, severe, and in some cases fatal. Allergic asthma is a major burden on patients, family, and society at large. It is associated with enormous health care costs, millions of lost workdays, school days, and might lead to a chronic, debilitating illness requiring long-term care. Despite a better grasp of the fundamental mechanisms of allergic asthma, a complete understanding of the pathogenesis, genetic susceptibility, and the increase in incidence and prevalence remains elusive. This review presents an overview and comparative analysis of experimental models of allergic asthma with a focus on developments in the past year. E-mail address: (M.M. Epstein) [email protected]. 1740-6757/$ ß 2004 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddmod.2004.11.009
The role of the immune system is to protect the host against foreign pathogens. However, sometimes the immune system reacts against non-infectious, otherwise harmless foreign proteins, i.e. pollen allergens. This type of adaptive (acquired) immune response begins with the uptake of protein allergens by antigen presenting cells (APC), processing of the protein into small peptide fragments in these cells, and then transport as a peptide: MHC class II complex to the cell surface (Fig. 1). The T cell receptor (TCR) on CD4+ T helper (Th) lymphocytes interacts with this complex resulting in T cell activation and differentiation. Subsequently, activated effector CD4+ Th cells ‘help’ B cells produce antibody and produce Th2-type (IL-4, IL-5, IL-13) cytokines that initiate recruitment of eosinophils, B cell production of IgE, and mucus hypersecretion by goblet cells in the airways. The reason why allergic responses are generated in certain individuals is unknown. Moreover, it is also not clear whether all ‘susceptible’ individuals develop allergic immune responses. Normally allergenic proteins do not elicit pathogenic immune responses in non-allergic individuals. There are three possible www.drugdiscoverytoday.com
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Figure 1. Allergen-specific adaptive immune response leading to allergic asthma. Allergen is taken up by antigen presenting (in this case the macrophage) cells, macrophages or DCs in the lungs. It is then processed into peptide fragments that are complexed within the cell with MHC class II and brought to the cell surface. The complex then interacts with the TCR on CD4+ T cells resulting in T cell activation, clonal expansion, alteration of cell surface molecule phenotype, and the production of cytokines. These cytokines then induce the differentiation and recruitment of eosinophils to the lungs and stimulate the production of IgE by B cells. The IgE produced binds allergen and crosslinks the Fce receptors on mast cells causing the release of vasoactive mediators. The combination of eosinophils, products from mast cell degranulation, and Th2 cytokines leads to smooth muscle contraction, mucus hypersecretion, edema, and an inflammatory infiltrate in the lungs. Repeated allergen exposure results in recurrence of disease and eventually might lead to permanent lung changes, termed airway remodeling, and lung destruction.
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explanations including: (1) non-susceptible individuals generate a non-allergic response, (2) they ignore the allergen, that is do not develop any type of response, and (3) they develop immunological tolerance (a process that causes immune unresponsiveness). Following sensitization, subsequent encounters with allergen induce more vigorous and rapid responses, which are initiated by Th2 memory cells (a feature of adaptive immunity) that reside in the lungs and by the release of vasoactive mediators by mast cells upon allergen-IgE crosslinking of cell surface Fce-receptors. The result is reversible airway obstruction caused by edema, increased mucus production, smooth muscle cell contraction, and eventual lung damage.
In vitro models of allergic asthma The adaptive and innate immune systems (T-lymphocytes, Blymphocytes, mast cells, eosinophils, macrophages, dendritic cells) and resident lung cells (respiratory epithelial, smooth muscle, and nerve cells) are all involved in allergic asthma. The relatively easy access to immune cells allows them to be isolated and purified from peripheral and cord blood, and respiratory tract cells can be isolated from sputum, nasal or bronchial washings, and biopsies, and subsequently cultured and evaluated for the expression of cell surface molecules, ability to proliferate, migrate, apoptose, and secrete. Typically, in vitro models utilize cultured single cell suspensions, however, newer models cultivate respiratory tract tissue explants.
Adaptive immunity Adaptive immunity relates to antigen-specific defense mechanisms that are designed to eliminate antigen and are maintained throughout life. The majority of in vitro models of allergic asthma involve cells of the adaptive immune system specifically Th2 cells from blood and the respiratory tract because they play a central role in the initiation and maintenance of immune responses to allergens. In vitro stimulation of T cells with allergen, mitogens, environmental agents, and antibodies directed against the TCR leads to cell proliferation, cytokine/chemokine production, up- or down-regulation of cell surface molecules, and migration [1–3]. Additionally, it is possible to transfect T cells with a specific molecule to determine its effect on cell function. A recent example of this approach involved the transfection of the T-bet transcription factor into isolated Th2 cells [4], which significantly altered cytokine production and migration properties of Th2 cells. To simplify analysis of peripheral T and B cells in vitro, it is possible to use whole blood [5], peripheral blood mononuclear cells (PBMC) [6–8], and cultured cord blood mononuclear cells [9]. The advantage of these approaches is the avoidance of further lymphocyte isolation and purification to determine function and phenotype. One of the latest advances in T cell biology involves the
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differentiation of T cells from embryonic stem cells in vitro [10]. It is tempting to speculate that new models of allergic asthma will evolve from in vitro differentiated stem cells from allergic individuals.
Innate immunity The cells of the innate and inflammatory immune system including lung macrophages eosinophils, basophils, mast cells, dendritic cells, and neutrophils are involved in initiation and perpetuation of allergic inflammation and probably lung tissue destruction. They can be cultured and studied in vitro. For instance, blood eosinophils purified and stimulated in vitro by allergen and cytokines, can be assessed for migration, reactive oxygen species production, survival and viability, and cell surface molecule expression [7,11,12]. In a recent investigation of neutrophil function, specific allergens stimulated degranulation and release of elastase, suggesting a potential role of neutrophils in allergic inflammation [13]. APC (macrophages and dendritic cells) play a crucial role in allergic immune responses. Although APC can be purified, isolated, and generated from CD34+ peripheral blood monocytes, their function can be evaluated indirectly by measuring the IL-12 production by PBMC in vitro. In a recent study, investigators correlated reduced PBMC-IL-12 production in the perinatal period with an increased propensity to develop allergic type immune responses and implied that APC function early in life has an impact on allergic susceptibility in later life [14]. Although most cells studied in vitro tend to be differentiated effector cells, early progenitors migrating from the bone marrow to tissue provide a constant source of effector cells during an allergic inflammatory response. Bone marrow-derived CD34+ stem cells from asthmatics stimulated with allergen [15] and stimulated cord blood CD34+ progenitor cells [16] can differentiate into eosinophils. In vitro analysis of cultured cord blood cells from offspring of allergic and non-allergic parents might predict the development of allergic disease [17]. In vitro evaluation of innate immune cells in the context of adaptive immunity is a promising approach for elucidating disease pathogenesis and target identification.
Resident lung cells Mucus hypersecretion, smooth muscle contractility, hypertrophy and hyperplasia, and secreted chemokines, cytokines and growth factors from epithelial cells play crucial roles in airway obstruction and allergic inflammation. Epithelial and muscle cells are harvested and cultured from biopsy specimens. The response of primary cultured airway smooth muscle cells can be studied in response to stimuli [18]. Additionally, bronchial epithelial cells can be cultured and assessed for chemokine and cytokine production [19]. Moreover, the effects of byproducts of inflammation, i.e. secreted cationic proteins from eosinophils on the epithelium can be studied in vitro [20]. Culturing primary epithelial cells from www.drugdiscoverytoday.com
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guinea pig trachea in the presence of IL-13 results in mature goblet cells and is a promising model to study mucus production [21]. An important advance in the study of bronchial epithelium is the production of human bioengineered bronchial equivalents [23]. Healthy or asthmatic human bronchial epithelial and fibroblastic cells from biopsy specimens cultured on a mesenchymal layer undergo differentiation resulting in ciliated and goblet cells in a pseudostratified epithelium that resembles human bronchi. Insights on the physiological and pathophysiological responses of bronchial epithelium and smooth muscle will enable the further understanding of the complex interaction between allergic response and respiratory tract during allergic asthma.
Tissue explants The advantages of studying human tissue explant models of allergic asthma rather than cell suspensions is that the microenvironment is maintained and it is possible to measure inflammatory cell function [23,24], mucus production [25], and bronchial constriction [26]. Remarkably, allergeninduced cytokine responses of bronchial explants and PBMC from patients with asthma lead to disparate results [23], suggesting that it is necessary to determine which model is more predictive. Another advance in the field of tissue explants involves the use of human precision-cut lung slices [26]. The method utilizes 250 mm slices resected from lungs, cultured with serum from allergic patients and allergen or IgE antibody, in the presence or absence of antagonists, and videomicroscopy to record bronchoconstriction of bronchioles.
In vivo models of allergic asthma Although in vitro assays are useful and have made significant advances in the elucidation of cellular events in allergic asthma, the complex mechanisms underlying sensitization, genetics, and pathophysiology necessitate in vivo studies that cannot be done in humans. Fortunately, animal models of allergic asthma mimic clinical disease (reviewed in [27]). There are numerous models in mice, rats, dogs, cats, monkeys, and sheep, but small rodent models tend to be more widely used, owing to lower research costs and the availability of a large variety of transgenic animals and reagents. Models are generated with many different antigens, in the
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presence or absence of adjuvant, and with differing protocols. They address the clinical course, long-term effects of disease, airway remodeling, immune tolerance, prenatal sensitization, and immunological memory. Furthermore, these models can be used for drug discovery and evaluation of treatment effectiveness during distinct stages of disease (Fig. 2).
Allergen-induced animal models The majority of in vivo models of allergic asthma are induced by immunizing and challenging the respiratory tract with antigen or allergen. Some examples of animal models in use include, Bermuda grass allergen-induced feline asthma [28], ovalbumin (OVA)-induced disease in guinea pigs [29,30], mice [27], and pollen-allergic dogs [31]. The models are characterized by eosinophilic lung inflammation, mucus hypersecretion, airway hyperresponsiveness (AHR), and elevated production of IgE. Although the most commonly used model is in mice, and by and large, reflects clinical disease, there are notable differences, which include a less crucial role of mast cells and IgE, and a transient rather than sustained increase in airway reactivity [27].
Spontaneous disease models An ideal animal model of allergic asthma is one that spontaneously develops disease. Two examples of naturally occurring allergic asthma models are Ascaris suum-allergic sheep [32] and flea-allergic dogs [8]. These models offer the opportunity to study disease initiation, genetic inheritance, and pathogenesis and have certain advantages; the animals can be followed long term because they can undergo repeated bronchoalveolar lavage (BAL), biopsy, AHR procedures, and can be examined using clinical radiological and other scanning techniques. Another spontaneous asthma model was generated recently in mice [33]. Investigators produced T-bet knockout mice that develop AHR, lung inflammation, and features of airway remodeling without allergen exposure. Although these mice provide novel experimental opportunities, it is necessary to determine whether this model represents allergic asthma or non-allergic asthma.
Humanized animal models To avoid the constraints of studying human cells in vitro and animal physiology, which does not exactly replicate the
Figure 2. Treatment intervention in an allergic asthma in vivo model. (a) BALB/c or C57BL/6 mice immunized with OVA (10 mg, without adjuvant) intraperitoneally (i.p.), 3 weeks apart generates a Th2 immune response and OVA-specific IgE. (b) One week later, mice are placed in a plastic box and aerosolized with a 1% solution of OVA with an ultrasonic nebulizer for 1 h twice daily for 2 consecutive days. Within 2 days (peaks on day 31), mice develop eosinophilic lung inflammation, mucus hypersecretion, and AHR. Mice recuperate from acute allergic asthma within 30 days of the last aerosol. (c) At anytime during their lifetime, after having recovered from acute disease, the mice will develop an allergic asthma relapse upon exposure to nebulized OVA. Underlying this immunological memory response is the life-long persistence of lung inflammatory infiltrates containing Th2 memory cells. This is a useful in vivo mouse model for drug discovery and for testing drug effectiveness. Treatment intervention is possible before sensitization (a), before the clinical manifestations of allergic asthma (b), and before disease relapse (c).
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Table 1. Comparison summary table In vitro models
In vivo models
In silico models
Pros
Easy access to cells Reduced complexity Utilization of human tissue Avoids the need for animals Individual cell responses can be measured Respiratory tract tissue explants now available
Complex system that more accurately resembles human disease Multiple variables can be evaluated Many animal models: including spontaneous and humanizedmouse models Able to study disease initiation, pathogenesis, susceptibility and in vivo treatment efficacy Small animals have shorter lifespans which enable studies on long-term disease progression Armamentarium of transgenic animals
Modeling of metabolic pathways, receptor–ligand drug interactions, treatment effectiveness Complex disease modeling is possible Improves speed of drug development Decreases potential drug adverse side effects, predicts response Might improve understanding of physiology Possible to generate virtual patients
Cons
Excludes microenvironment influences and might be misleading
Animal models are not exact replicas of human disease
Explants exclude macroenvironmental influences Long-term effects cannot be observed Bronchial biopsies are not trivial, raise ethical concerns
Transgenic knockout mice generated using different techniques might lead to disparate results Larger animal studies are expensive, less available, longer lifespans
Simulation models reflect what is known; if the mechanism is not entirely clear, the model might be erroneous
Evaluation of cell physiology, function, viability, phenotype, and responses to stimulators and inhibitors
Evaluation of disease initiation, host susceptibility Perform pre-clinical treatment studies Evaluation of chronic disease and lung remodeling
Best use of model
References
[1–25]
Ethical considerations regarding the use of animals in research
[26–36]
clinical situation; several laboratories have established human–mouse chimeric models. PBMC from allergic patients injected into B and T lymphocyte deficient severe combined immunodeficiency (SCID) mice produce the human-SCID (hu-SCID) mouse model. Although most cells belong to the recipient mice, many cells, including lymphocytes and dendritic cells are of human origin. When hu-SCID mice are aerosolized with the offending allergen, they develop allergic asthma characterized by human IgE, pulmonary inflammation, AHR [34,35], and airways and lungs infiltrated with human T lymphocytes [36] and dendritic cells [37]. Although this model offers advantages, it is unclear whether human cells function in mice exactly as they do in humans. In another human–mouse model, investigators utilized a clinically relevant allergen, short ragweed, Ambrosia artemisiifolia, in transgenic mice expressing human MHC class II (DQ8;HLADQA1*0301 and HLA-DQB1*0302). This model offers the opportunity to evaluate human HLA class II influences and the roles of other T cell and APC molecules, such as, CD28/B7 in allergic asthma [38].
In silico models of allergic asthma Computer simulations used to generate virtual ligand–receptor interactions, cells, respiratory tracts, and patients are the newest wave in modeling allergic asthma. 392
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Simulations of cell responsiveness, disease induction, factors involved in generating chronic disease, prediction of drug treatment efficacy, adverse side effects [37,38]
Virtual drug discovery In silico screening to identify novel drugs might become a useful tool. An example of this approach in allergic asthma is a computational screening of ligand–receptor interactions based on 3D structure of the X-ray analysis of a particular binding site. This creates a 3D ‘pharmacophore’ model, which generates a pattern of spatial and chemical constraints of the ligand. This approach was used to produce non-peptidic antagonists of VLA-4, an adhesion molecule involved in cell–cell interactions [32]. The compounds identified from a virtual library were chosen on the basis of an IC50 comparable to the original ligand. The synthesized compounds were then tested in an allergic sheep model of asthma. This is an example of an integrative approach of in silico, pharmacological, and in vivo models.
Virtual organs A variety of computer simulations of lungs have led to the socalled, ‘lung physiome’ or ‘virtual lung’. These simulations provide physiological measurements of healthy and asthmatic lungs, and can be used for testing drug efficacy. One example is the simulation of a 3D human respiratory tract using MRI and SPECT images from lungs and thorax. The virtual respiratory tract enables researchers to evaluate lumen diameter based on bronchoconstriction, inflammation, and
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mucosal thickness and has been used to predict the efficiency of aerosol drug delivery in healthy versus asthmatic subjects [39].
Virtual patients The virtual allergic asthmatic promises to be the next generation of in silico model. One example is the virtual rat lung model developed to determine the effects of environmental pollution on lung function (www.pnl.gov). A second example is a computer-based mathematical model of airway structure and function, inflammation, and immune responses to create virtual asthmatic patients (Entelos1 Asthma PhysioLabTM) [40] by the company, Entelos Inc., Menlos, CA, USA (www.entelos.com). A major aim of virtual patient models is to reduce the costs of getting new drugs to the market. However, there is growing interest for these models to be used to study disease pathogenesis. An example is the physiome project (www.physiome.org), which is dedicated to the generation of databases from existing knowledge to produce physiomes or virtual computer-generated models of humans and other animals to study pathophysiology.
Model comparison There is no ‘best’ model of allergic asthma. Taken together, in vitro, in vivo, and in silico models of allergic asthma are constantly accumulating information to increase the knowledge about initiation and perpetuation of disease, genetic susceptibility, pathogenesis, effectiveness of therapy, and to find better drugs. Individually, the models have strengths and weaknesses that need to be acknowledged (Table 1). Cellular in vitro assays exclude the effect of microenvironment influences and cannot recapitulate disease complexity. By contrast, tissue explant models include microenviroment but exclude macroenvironment influences. Additionally, the discrepancy between PBMC and explant results suggests that old questions need to be revisited. Another concern is that in vitro lung tissue models require bronchial biopsy, which is a safe but not trivial procedure. Moreover, it is rarely clinically indicated for asthmatics and thus, raises ethical considerations. In vivo models are necessary because of the complexity of disease. Furthermore, cell and explant assays cannot completely reproduce the conditions necessary for understanding allergic asthma. The allergen-induced and spontaneous animal models are important, but they are not exact replicas of human disease and thus, are not ideal. Humanized-mouse models combine the advantages of both human cells and in situ microenvironment, but have not excluded xenogeneic influences. Additionally, heavy reliance on in vivo models, especially genetically mutated knockout mice might be unwise. Recently, two laboratories established mice lacking eosinophils to study their role in allergic asthma [41,42]. They used different experimental strategies and generated conflicting results. The reason for these disparate observa-
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tions is not known but is possibly owing to mouse strain differences or related to the approach used to delete the eosinophils. Although these findings have not definitively elucidated a role for eosinophils in allergic asthma, they illustrate that transgenic in vivo models must be interpreted cautiously. In silico models have potential in allergic asthma. Modeling of ligand–receptor interactions offer a valuable approach for drug design and organ modeling has important implications for lung physiology and drug discovery. An exciting possibility is comparative studies of virtual healthy and asthmatic patients. However, a major problem is that the generation of simulated humans is limited by the extent of existing knowledge. The model can only be as accurate as the database.
Conclusions Many of the advances in allergic asthma have been the result of an interrelationship between in vitro and in vivo modeling. Importantly, in vitro observations are confirmed in vivo and vice versa. This establishes a phenomenon that might be followed by the generation of a transgenic knock in or knock out in vivo model. The combination of data from all models might then be used as the basis for clinical and epidemiological studies in humans. The hope is that in silico modeling and the generation of virtual patients will eventually reduce the need for clinical studies. In summary, a complementary approach incorporating experimental observations from in vitro, in vivo, and in silico models is necessary. A daunting task will be the gathering, evaluating, integrating, and ultimately synthesizing all available data.
Acknowledgements I would like to thank B. Mittleman, P. Stuetz and O. Hoffmann for their critical reading of the manuscript.
Outstanding issues
When and how is allergic asthma initiated? Who is susceptible to allergic disease? What factors influence disease susceptibility? What is the effect of repeated disease exacerbations on lung function? What role do environmental factors (other than allergen) play in allergic asthma? Why is incidence and prevalence increasing in the industrialized world?
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