INTRODUCTION

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Keywords: airway remodeling; asthma; transgenic modeling; interleukin 11; interleukin 13

Airway remodeling is an inclusive term that refers to the noninflammatory alterations in structural cells and tissues in the asthmatic versus the normal airway. Major features of the remodeling response include airway wall thickening, fibrosis in the subepithelial regions and nearby interstitia of the airways, myocyte hypertrophy and hyperplasia, myofibroblast hyperplasia, and mucous metaplasia. Vascular abnormalities have also been described. Our present concepts of disease pathogenesis suggest that remodeling is due to the chronic inflammation that is characteristic of the asthmatic airway. They also suggest that the various features of the remodeling response contribute to clinical and pathophysiologic features of the asthmatic disorder. The latter assumptions are based on studies that suggest that airway remodeling contributes to the pathogenesis of airway hyperresponsiveness, asthma severity and the generation of incompletely reversible or fixed airway obstruction. It is important to point out, however, that these concepts have not been proven and that many important issues in airway remodeling have not been addressed with an adequate level of scientific certainty. Specifically, we do not know how common airway remodeling is in asthmatic populations, why only some patients develop airway remodeling, and what contribution each facet of the remodeling response makes to the symptoms, physiology, and natural history of the disorder. We are also limited in our knowledge of the reversibility of the features of the remodeling response and what the cellular and molecular events are that are involved in their pathogenesis. Last, we do not know whether the remodeling response represents a healing and repair response and/or aspects of disease pathogenesis.

	CONSTITUTIVE AND INDUCIBLE OVEREXPRESSION TRANSGENIC MODELING OF IL-11 AND IL-13

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Investigations of the pathogenesis of airway remodeling in asthma and other disorders, in the postgenome era, will undoubtedly focus on the paradigm of "genes without function." Specifically, the genome projects, research approaches such as differential display and investigations using microarray approaches have and will continue to provide us with large amounts of primary genetic sequence information and knowledge about the dysregulation of genes in disease. One of the greatest challenges for biologic researchers over the next decade or so will be to decipher the role that these genes play, alone and in combination, in normal homeostasis and pathologic disease states. To begin to obtain this sort of information, we have used overexpression transgenic modeling to define the chronic effector functions of lung-relevant cytokines in vivo. Two different overexpression transgenic systems have been validated and utilized that allow for constitutive and inducible overexpression transgenic modeling.

Constitutive Overexpression Modeling

In these investigations, a lung-specific promoter is used to target the transgene in question to the lung. The Clara cell 10-kDa (CC10) and surfactant apoprotein C (SP-C) promoters are used most frequently. The CC10 promoter expresses the transgene in airway Clara cells and is useful when airway phenomena are being investigated. Its efficacy in these transgenic systems is based on a convenient species difference, with up to approximately 40 and 3% of airway epithelial cells being Clara cells in the mouse and human, respectively. In these systems, CC10 initiates gene transcription in utero and drives transgene expression in a constitutive fashion throughout the life of the transgenic animal.

Inducible Overexpression Transgenic Modeling

The constitutive overexpression system described above has a number of limitations. Specifically, it is unable to accurately model waxing and waning disease processes such as asthma. In addition, it does not allow development-dependent and adult-onset responses to be differentiated. To allow waxing and waning responses and the influences of lung development to be adequately assessed, we have developed a dual transgenic inducible overexpression system that allows the investigator to control the timing of transgene expression (1). It is based on the generation of dual transgenic mice. Construct 1 in this system contains the CC10 promoter driving the expression of the reverse tetracycline trans-activator (rtTA) in a lung-specific fashion. The reverse tetracycline trans-activator is a fusion protein made up of a mutated tet Repressor (tet-R) protein and the herpesvirus VP-16 trans-activator. Tet repressor proteins bind with high avidity to tetracycline operator (tet-O) sites. The tet-R in these constructs has been mutated in a fashion that allows it to bind to tet-O sites only in the presence of doxycycline (dox). VP-16 is a powerful activator of gene transcription. The second construct contains multimers of the tet-O, a minimal promoter, and the transgene of interest. Under normal circumstances, the CC10 promoter targets rtTA to the lung. If the animal is receiving dox (either in its drinking water or as a subcutaneous pellet), the rtet-R binds to the tet-O sequences, allowing VP-16 to activate the transgene. In the absence of dox, rtTA is produced but does not bind or binds weakly to the tet-O and gene transcription is not initiated in a major fashion. Thus, this transgenic system allows the investigator to induce high level transgene expression by adding dox to the animal's drinking water.

Transgenic Modeling of IL-11

Interleukin 11 (IL-11) was discovered as an activity in an "IL-6" plasmacytoma proliferation bioassay that could not be neutralized with antibodies against IL-6. It has subsequently been classified as a member of the IL-6-type cytokine family on the basis of the partially overlapping effector profiles of IL-6, IL-11, and other members of this family and the common use of the gp130 signaling  subunit in the multimeric IL-6 and IL-11 receptor complexes. The majority of the early investigations of IL-11 focused on its role in the regulation of platelet production. Thus, from the point of view of visceral organs, IL-11 fits the paradigm of a gene without a known biologic function.

We initiated our studies of IL-11 in the lung by determining whether IL-11 could be produced by lung cells and tissues. These studies demonstrated that lung epithelial cells, fibroblasts, and smooth muscle cells produce IL-11 in response to a variety of stimuli including transforming growth factor  (TGF-) moieties, IL-1, respiratory viruses (rhinovirus, parainfluenza virus type III, and respiratory syncytial virus), histamine, and eosinophil major basic protein (2). To understand what IL-11 might be doing in these settings we needed to define the effector functions of IL-11 in respiratory structures. This was accomplished by generating transgenic mice in which the CC10 promoter was used to constitutively overexpress IL-11 in the murine lung. Multiple lines of CC10-IL-11 animals were isolated and characterized. When compared with transgene(-) littermate controls, histologic analysis of transgenic mice demonstrated (1) peribronchiolar nodular collections of mononuclear cells including B cells and T cells, (2) enlarged alveoli, and (3) airway wall thickening. Mallory's trichrome evaluations demonstrated subepithelial and adventitial tissue fibrosis and immunohistochemistry (IHC) revealed the enhanced deposition of types I and III but not type IV collagen. -Smooth muscle actin IHC also revealed the enhanced accumulation of -smooth muscle actin(+) cells in the subepithelial region and ultrastructural analysis demonstrated increased numbers of fibroblasts, myofibroblasts, and myocytes in this location. Pulmonary function evaluation of these mice revealed baseline airway obstruction. They also revealed airway hyperresponsiveness on methacholine challenge (8).

It is well known that the normal mouse is born with large alveolar sacs that are not true alveoli. True alveoli form in these animals during the first month of life via a developmental process that includes alveolar septation. We thus reasoned that the enlarged alveoli that were seen in these animals could be the result of a developmental abnormality or the result of the ability of IL-11 to induce alveolar degradation (as seen in adult emphysema). To investigate this process, we used the CC10-rtTA system described above to selectively express IL-11 in the adult lung. When IL-11 gene expression was delayed until the animal was 1 month old, subepithelial fibrosis and inflammatory nodules were noted. Interestingly, normal-sized alveoli were consistently obtained (1). This demonstrates that the alveolar abnormality that is seen in these animals is due, at least in part, to the ability of IL-11 to alter alveolar development. It also demonstrates that IL-11 can cause airway remodeling in the absence of major alveolar abnormalities. This is analogous to the situation that is noted most commonly in remodeled asthmatic airways.

Transgenic Modeling of IL-13

Helper T cell type 2 (Th2) cytokines are known to play a major role in the pathogenesis of asthma. To further our understanding of the chronic in vivo effector functions of these cytokines, similar constitutive overexpression transgenic approaches were used to model the effects of IL-13 in the lung. Two-month-old transgene(+) mice manifest an interesting phenotype that includes prominent peribronchial inflammation with enhanced numbers of macrophages, lymphocytes, and eosinophils; epithelial hypertrophy; impressive mucous metaplasia with the enhanced expression of a variety of mucin genes including Muc-5ac; and subepithelial fibrosis (9). Enhanced tissue hyaluronic acid accumulation was also documented via immunohistochemical analysis and exaggerated levels of eotaxin protein and mRNA were noted in bronchoalveolar lavage (BAL) fluids and whole lung RNA using enzyme-linked immunosorbent assay (ELISA) and Northern blot analyses. Physiologic evaluation of these mice demonstrated modest degrees of airway obstruction at baseline and impressive airway hyperresponsiveness on methacholine challenge.

	IL-11 AND IL-13 IN HUMAN ASTHMA

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Like all experimental modeling systems, transgenic mice have their advantages and limitations. They can, however, be most appropriately considered furry, four-legged hypothesis generators. They provide insights into biologic paradigms that may be important in the pathogenesis of human disease. This allows rational hypotheses to be generated that can then be tested in human patients and samples. The relevance of the findings in the IL-13 overexpression transgenic mice to human asthma are easily appreciated because a large number of studies have demonstrated the exaggerated production of IL-13 in humans with the asthmatic diathesis (10). Until recently, however, knowledge of IL-11 expression in asthma had not been obtained. In collaborative studies with Q. Hamid and E. Minshall (11), in situ hybridization and IHC were used to compare the levels of expression of IL-11 in lungs from normal subjects and subjects with mild, moderate, and severe asthma. These analyses did not reveal significant difference in the expression and/or accumulation of IL-11 in tissues from normal subjects and patients with mild asthma. In contrast, in situ hybridization and IHC revealed impressive levels of IL-11 mRNA and protein in tissues from patients with severe asthma. Intermediate levels were seen in patients with moderately severe asthma. In all cases, IL-11 mRNA and protein were noted in airway epithelial cells and EG2(+) eosinophils. Interestingly, the epithelial IL-11 index in these patients correlated inversely with their forced expiratory volume in 1 s (FEV₁) and directly with the degree of severity of their disease (higher levels of IL-11 were seen in patients with more severe disease) (11).

	IL-11 IN HEALING AND REPAIR

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The studies noted above demonstrate that IL-11 can be produced by a variety of lung cells in response to a variety of stimuli. They also demonstrate that IL-11 induces tissue fibrosis and is found in exaggerated quantities in airway biopsies from patients with severe asthma. Studies were next undertaken to determine whether IL-11 production represented a feature of disease pathogenesis and/or a manifestation of airway healing and repair. We reasoned that cytokines that are involved in healing and repair would simultaneously induce tissue fibrosis and the resolution of tissue inflammation (as seen with TGF-). In contrast, we reasoned that cytokines that are involved in inducing a tissue fibrotic response in an attempt to preserve organ integrity in the face of ongoing injury would induce this fibrotic response without necessarily inhibiting ongoing inflammatory responses. To gain insight into the role(s) of the IL-11 that is produced in the asthmatic airway we characterized the effects of IL-11 in a standard murine asthma model that involves systemic sensitization and aerosol challenge with ovalbumin. This was done by comparing the ovalbumin-induced responses in IL-11 transgene(+) mice (CC10-IL-11 mice) and wild-type transgene(-) littermate controls. Lung-targeted IL-11 did not alter the ability of transgene(+) and transgene(-) mice to be sensitized after ovalbumin exposure. IL-11 did, however, have a profound inhibitory effect on antigen-induced inflammatory responses in the lung. This inhibitory response was associated with marked diminution in eosinophil recruitment, Th2 cell accumulation, and Th2 cytokine production (12). Interestingly, IL-11 did not alter the ovalbumin-induced mucous metaplasia. These studies demonstrate that IL-11 inhibits antigen-induced inflammation while inducing local tissue fibrosis. This suggests that IL-11 acts as a healing cytokine in the asthmatic airway and other tissue locales.

	TETRACYCLINE-CONTROLLED TRANSCRIPTIONAL SILENCER ON/OFF MODELING OF THE NATURAL HISTORY OF AIRWAY REMODELING

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One of the major unanswered questions as regards the remodeling response in the airway relates to the reversibility versus lack of reversibility of the phenotypic alterations that are noted. The inducible transgenic system noted above has been proposed as a system in which transgenes can be expressed in pulmonary tissues, remodeling responses generated, and then gene expression turned down. This would allow the permanence of the phenotypic alteration to be assessed over time. This system can, however, have significant levels of basal transgene leak. To address this leak, a new, triple transgenic system has been developed (13). It contains constructs 1 and 2 involved in the CC10-rtTA inducible transgenic system described above. It also contains a construct in which the CC10 promoter drives the expression of the tetracycline-controlled transcriptional silencer (tTS) (Figure 1). The tetracycline-controlled transcriptional silencer is a fusion protein made up of a wild-type tet-R and the KRAB-AB domain of the Kid-1 protein. The tet-R in this construct binds to the tet-O in the absence of dox and releases from the tet-O when dox is administered. The KRAB domain is a powerful transcriptional inhibitor that after local binding inhibits promoter activity. In this system, triple transgenic mice are generated. In the absence of dox, both tTS and rtTA are produced in a lung-specific fashion. The tTS binds to the tet-O, inhibiting basal transgene expression. Once dox is added, the tTS is released and the rtTA binds to the tet-O, activating gene transcription. Experiments using this triple transgenic system have demonstrated that, in CC10-rtTA-IL-13 transgenic mice, basal transgene leak is totally abrogated and that dox-induced transgene activation is unaltered. Thus, the addition of tTS to our transgenic system has changed it from one in which there is low-level and high-level gene expression to one in which gene expression can be truly "on" or "off." We believe this system will prove useful in defining the reversibility of transgene-induced phenotypic alterations and remodeling responses in the lung.

View larger version (27K): [in this window] [in a new window]   Figure 1.   Schematic illustration of tTS "on/off" transgenic modeling system. Definition of abbreviations: Pcmv = cytomegalovirus promoter; rtTA = reverse tetracycline trans-activator; Tet-OBP = tetracycline operator-binding protein; tTS = tetracyline-controlled transcriptional silencer.

	SUMMARY

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Students of lung biology frequently deal with diseases (such as asthma) that are characterized by chronic tissue inflammation and disease manifestations that evolve slowly over time. Unfortunately, our present investigative approaches are much better suited to characterizing the pathogenesis of acute, in contrast to chronic, pulmonary responses. As a result, the cellular and molecular events that are involved in disease pathogenesis are more difficult to define in these chronic disorders. To address these difficulties, transgenic approaches have been developed that allow genes to be selectively expressed in the lung. These overexpression transgenic approaches have proven to be useful tools that allow investigators to characterize the chronic effector functions of proteins in the airway and, in turn, define biologic paradigms that may be operative in humans with chronic pulmonary disorders. The power of the transgenic approach has been enhanced by improvements that allow transgene expression to be externally controlled. This allows waxing and waning diseases and development-dependent diseases to be modeled with enhanced fidelity. In addition, tTS-based systems may now allow investigators to turn gene expression "off" in vivo, allowing the reversibility of phenotypic responses to be defined. Efforts that use overexpression transgenic approaches to generate hypotheses and human-based studies to test these hypotheses will provide important information upon which future therapeutic interventions can be based.