New insights into the pathogenesis of asthma (original) (raw)
In contrast to the vast majority of injury and repair responses in the lung and other organs, asthmatic inflammation frequently starts in childhood and persists throughout the life of the afflicted individual. In addition, physicians treating asthmatics invariably find themselves attempting to deal with manifestations of established disease, often in the setting of a disease exacerbation. Surprisingly, the model systems that have been used most frequently in studies designed to understand asthma pathogenesis have not appropriately taken these issues into account. Instead, the most commonly employed modeling systems evaluate the acute responses that are elicited in the lung after normal animals are sensitized to and then challenged with an aeroallergen. The asthma-like inflammation and physiologic dysregulation that are seen in these models are an end result of the cellular and molecular events involved in sensitization, Th2 cell development, Th2 cytokine elaboration, and the activation of Th2 cytokine effector pathways. Interventions that inhibit any of these steps can appear to have a beneficial effect on the asthma-relevant readouts that are employed. However, since it is likely that antigen sensitization, Th2 cell development, and Th2 cytokine elaboration have already occurred in patients with established disease and/or a disease exacerbation, interventions at these sites will likely be less than useful therapeutically. In contrast, interventions that regulate Th2 cytokine effector pathways are attractive as therapies. Until recently, modeling systems that allowed the inflammatory and remodeling effects of chronically elaborated Th2 cytokines to be selectively evaluated did not exist. Overexpression-transgenic methodology has, however, powerfully addressed this issue.
The standard approaches used to generate overexpression-transgenic mice are illustrated in Figure 3. First, a DNA construct is prepared that contains the gene that the investigator wishes to express and a promoter to drive the expression of this gene in the desired organ and/or tissue (Figure 3a). If temporally regulated gene expression is desired, recent advances in transgenic methodology that involve the generation of double- and triple-transgenic animals allow the transgene to be selectively turned on or off at any time during the life of the animal (6, 7). When asthma-relevant questions are being asked, the Clara cell 10-kDa protein (CC10) promoter is used to target gene expression, because it is selectively expressed by the Clara cells that make up 40% of the epithelium of the murine airway. To generate transgenic mice, male and female mice are allowed to mate, and the fertilized eggs are washed out of the female’s oviduct. The desired DNA construct is then directly microinjected into the pronuclei of these eggs, and the eggs are placed in the uterus of a pseudopregnant mouse. A pseudopregnant mouse is a female that has been mated with a vasectomized male. She is behaving, from a hormonal perspective, as if she is pregnant and becomes pregnant when the fertilized eggs are deposited in her uterus. She subsequently carries to term, delivering a litter of pups, some of which have the transgene randomly integrated into their genome, others of which do not (Figure 3b). Transgene-positive and -negative mice can be differentiated by extracting DNA from tail biopsies from the pups and determining whether the transgene is present by Southern or PCR analysis. Thus, an outstanding experimental system is established where one can compare the phenotypes of mice born to the same mother, on the same day, that are exposed to the same environment and differ only in the one gene that was inserted. The power of this approach can be easily appreciated in studies designed to define the effects of IL-13 in the asthmatic airway (8).
Generation of transgenic mice. To express a transgene in vivo, the investigator first makes a construct containing the transgene being evaluated. A typical construct is illustrated in a. It contains a promoter that targets the transgene to the desired organ, the transgene being expressed, and intronic and polyadenylation sequences that ensure the proper processing of the mRNA transcripts that are produced. The methodology for generating transgenic mice is illustrated in b. Fertilized eggs are washed out of the oviducts of female mice. They are then microinjected under direct visualization and implanted into the uterus of pseudopregnant female mice. The genotype of the pups that are produced is evaluated in tail biopsy–derived DNA using PCR reactions or Southern blot evaluations.
IL-13 is the product of a gene on chromosome 5 at q31, a site that has been repeatedly implicated in genetic studies looking for the genes involved in the asthmatic diathesis. It was originally discovered as an IL-4–like molecule and was presumed to have an effector profile identical to that of IL-4. It has since become clear that IL-13 and IL-4 differ in their effector properties, with IL-4 and IL-13 playing more prominent roles in the initiation and the effector phases of Th2 inflammation, respectively.
The effector functions of IL-13 were defined and clarified using overexpression-transgenic modeling systems. These studies demonstrated that IL-13 is a potent inducer of an eosinophil-, macrophage-, and lymphocyte-rich inflammatory response, airway fibrosis, mucus metaplasia, and airway hyperresponsiveness (8) (Figure 4). These studies also demonstrated that other Th2 cytokines, such as IL-9, mediate their effects in the lung via their ability to induce IL-13 (9), suggesting that IL-13 may be a final common pathway for Th2-mediated inflammatory responses. Importantly, these transgenic systems were also manipulated to define the mechanisms by which IL-13 generates these critical tissue responses. This was done using standard methods of quantitating mRNA and gene chip methodology to define the genes that are regulated by IL-13 in lungs from transgenic mice. This was followed by a variety of manipulations that characterized the contributions of specific genes to the pathogenesis of the IL-13 phenotype. One such manipulation was the use of neutralizing antibodies against the gene products in question. Another was the breeding of the IL-13 transgenic mice with mice with null mutations of selected downstream genes, followed by characterization of the effects of the transgene in mice that were sufficient or deficient in the downstream gene in question (Figure 5). As can be seen in Figure 6, these studies have provided impressive insights into the mechanisms of IL-13–induced inflammation and tissue fibrosis. The inflammatory response is mediated by the ability of IL-13 to stimulate the elaboration of chemotactic cytokines called chemokines and proteolytic enzymes called matrix metalloproteinases (MMPs) (10, 11). These studies demonstrate that chemokine receptor 2 (CCR2), MMP-9, and MMP-12 play key roles in these responses (10, 11). They also demonstrate that the fibrotic response results from the ability of IL-13 to stimulate the production and activation of the fibrogenic cytokine TGF-β1, and that TGF-β1 is activated via an MMP-9– and plasmin-dependent pathway in this setting (12). These studies provide a road map that defines the pathways that IL-13 uses to generate tissue inflammatory and remodeling responses. They also highlight target genes against which therapies can be directed to control selected aspects of the IL-13–induced tissue response.
Demonstration of the effects of transgenic IL-13 on airway fibrosis and mucus metaplasia. (a) Trichrome stains are used to compare the amount of blue-staining collagen around airways from transgene-negative mice (left) and transgene-positive mice (right). (b) Alcian blue stains are used to demonstrate mucus accumulation in airways from transgene-negative mice (left) and transgene-positive mice (right). Mucus is blue in this evaluation.
Use of null mutant (knockout) mice to define the pathways that transgenes use to generate disease-relevant phenotypes. In these experiments, transgenic mice with a disease-relevant phenotype (for example, fibrosis or inflammation) are mated with mice that have a null mutation of a downstream gene that is believed to play an important role in the generation of this phenotype. Transgene-positive (TG[+]) and transgene-negative (TG[–]) mice are generated that have normal downstream genes (+/+), are heterozygote knockout at the downstream gene in question (+/–), or are null-mutant for the downstream gene in question (–/–). The presence and intensity of the phenotypes of these mice are then compared. These comparisons allow an investigator to define the role(s) that this downstream gene plays in the generation of the pathologic response.
Mechanisms of IL-13–induced phenotype generation. IL-13 binds to the IL-13 receptor complex made up of IL-4 receptor α (IL-4Rα) and IL-13 receptor α1 (IL-13Rα1). IL-13 also binds to IL-13Rα2, which is a decoy receptor that inhibits IL-13 responses. After binding to the IL-13 receptor complex, IL-13 activates STAT-6 signal transduction pathways. Pathways that involve chemokines, the chemokine receptor CCR2, MMPs, urinary plasminogen activator (UPA), TGF-β1, VEGF, and/or adenosine are then activated, and inflammation, fibrosis, blood vessel alterations, and mucus responses are generated. Each of these pathways is a site against which therapeutic agents can be directed.