Transgenic and gene-targeted mice as models for chronic obstructive pulmonary disease

Animal models have played an important role in the understanding of the pathogenesis of chronic obstructive pulmonary disease (COPD). Animal models figured prominently in the origin of the elastase–antielastase hypothesis in the 1960s, when Gross et al. 1 found that instillation of elastases led to emphysema in rodents. With the advent of genetic engineering, the capacity is now available to specifically overexpress and delete individual gene products in mice, allowing for highly controlled experiments in mammals. These techniques provide opportunities to dissect disease pathways in vivo. However, the applicability of findings to human COPD depends upon several factors, including the disease model, and similarities in mouse structure and function between species. The present author’s hypothesis is that general mechanisms are likely to be well conserved, but important details may differ considerably; hence, care in translation from mouse to humans is required.

TRANSGENIC MICE

To generate transgenic mice, the cDNA of interest is linked to a promoter (and a polyA tail), which is often lung-specific, and the DNA or “transgene” is microinjected into pronuclei of fertilised eggs to randomly integrate genetic material into the genome 2. Upon placement of eggs into the oviduct, offspring may then express the transgene. Newer developments allow conditional (cell-specific) and inducible expression. Inducible expression avoids the developmental expression of the transgene. This is particularly important in the study of COPD, to avoid confusion between destruction and enlargement of airspaces that define emphysema, and enlarged airspaces due to failed septation impairing alveogenesis during lung development (fig. 1⇓). Lineage-specific promoters (or knock-in technology) linked to colour markers that tag particular groups of cells can also be used. These techniques will become very useful in the identification of specific stem cells in the future.

Transgenic mice have been used in COPD to overexpress a particular gene product to obtain a COPD phenotype, usually airspace enlargement. Hence, this technique will allow the deduction that “if” this particular gene is expressed at high levels, it “could” lead to aspects of COPD. If it is known that the protein is in fact elevated in COPD, this provides strong evidence that it contributes to pathology. There are now many examples in the literature of transgenic mice that have contributed to the understanding of COPD.

One of the earliest applications of transgenic technology to COPD was the overexpression of collagenase-1 (matrix metalloproteinase (MMP)-1) resulting in airspace enlargement 3. This challenged the elastase–antielastase hypothesis, thereby placing collagen squarely in the picture. A limitation of the study 3 is that while expression was lung specific in some lines (despite being driven by the haptoglobin promoter), it was not inducible. However, the authors did not detect expression of collagenase in some lines during early development. The role of collagen in COPD is complex. Clearly, destruction of an alveolar unit requires loss of all cells and extracellular matrix including collagen; however, total collagen content in COPD is actually increased, with submucosal airway fibrosis likely to contribute to airflow obstruction. Polymorphisms in the collagenase promoter that cause higher levels of collagenase expression actually correlated with protection against decline in forced expiratory volume in one second in the large lung health study population 4. However, mice do not possess MMP-1 but do express the two other MMP collagenases (MMP-8, neutrophil collagenase or collagenase-2, and MMP-13 collagenase-3), which compensate for lack of collagenase-1 in rodents.

Overexpression of interleukin (IL)-13 5 and interferon (IFN)-γ 6 represent two important examples of inducible, conditional transgenes leading to emphysema in adult mice. Overexpression of the T-helper cell (Th)2 cytokine IL-13 resulted in inflammation and lung destruction that was metallo- (MMP-9 and MMP-12) and cysteine proteinase-dependent. These mice also exhibit airway remodelling with goblet cell hypertrophy and subepithelial collagen deposition. MMP-9-mediated transforming growth factor (TGF)-β activation was responsible for collagen remodelling in this model 7. Whether IL-13 is overexpressed in human COPD is currently under investigation. This mouse also supports the “Dutch hypothesis”, which states that asthma and COPD have common underlying mechanisms. Overexpression of the Th1 cytokine IFN-γ also resulted in inflammation and proteinase-dependent emphysema. Compared with the IL-13 transgenic mouse, the inflammatory component with IFN-γ was more subtle, apoptosis was prominent, and there was no associated airway pathology. These are just two examples that demonstrate the complexity of inflammatory networks and how unexpected findings in animal models have led to the search for new potential mediators in human disease.