Thirty-five years ago Gross instilled papain into experimental animals resulting in emphysema (1). This finding, combined with the clinical observation by Laurell and Erickson that patients with deficiency of α-1-antitrypsin (α-1AT) were at increased risk of emphysema (23), formed the scientific basis for the elastase:antielastase hypothesis for the pathogenesis of emphysema. Today, this remains the prevailing theory for the development of emphysema, and animal models of chronic obstructive pulmonary disease (COPD) remain a critical experimental tool. In this issue, Suga and colleagues report that the absence of klotho results in air-space enlargement in mice (2).
A more comprehensive statement of the elastase:antielastase hypothesis is that cigarette smoking causes inflammatory-cell recruitment into the lung, activating and releasing proteinases in excess of inhibitors in interstitial microenvironments. This, coupled with abnormal alveolar and matrix repair, results in emphysema. We are beginning to define the contribution of specific cells and proteinases, but we have yet to understand many crucial issues such as sites and mechanisms of airflow obstruction, as well as the molecular basis for abnormal repair. Why only a minority of cigarette smokers present with clinically significant emphysema remains a fascinating enigma, the study of which could lead to insight into mechanisms and cure.
Since Gross' initial experiments, investigators have instilled a variety of proteinases into the lungs of many small and large animals. A common feature is that administration of elastolytic enzymes, including pancreatic elastase, neutrophil elastase, and proteinase 3, result in air-space enlargement (3). Pancreatic elastase produces the most consistent and impressive airspace enlargement. Instillation of nonelastolytic enzymes, such as bacterial collagenase, does not cause emphysema. Overexpression of proteinases, either by simple intratracheal instillation or more modern transgenic methods, can determine whether an enzyme has the capacity to cause emphysema (if applied to mature, fully developed lungs). However, these models cannot identify which proteinases are involved in the pathogenesis of emphysema associated with cigarette smoking, nor can they be used to decipher events upstream of proteinase release. Moreover, cigarette smoke exposure may cause a variety of other abnormalities not observed with simple overexpression of a proteinase. Nevertheless, the elastase model remains in use because of its relative simplicity and the fact that it allows for first-order approximation for study of downstream events, particularly alveolar repair. For example, elastase instillation has recently been used to demonstrate that retinoic acid has the capacity to promote alveolarization and lung repair in adult male rats (4).
A variety of chemicals and irritants have been used in experimental animals to induce inflammation and emphysema, including lipopolysaccharides (LPS), cadmium chloride, nitrogen dioxide, inorganic dusts, and ozone. Results from these models are reviewed elsewhere (3). In short, all have contributed to our knowledge of lung injury, but none have replicated exposure to cigarette smoke as a model for authentic COPD. A variety of animals have been exposed to cigarette smoke over the years, including dogs, rabbits, guinea pigs, and rodents (3, 5). Recent focus has been on the mouse because it provides unique opportunities for genetic manipulation. Other advantages of the mouse include great knowledge of mouse biology, abundant probes, rapid breeding, large litter sizes, small size (good for dosing expensive drugs; bad for surgical models), and relatively cheap housing.
We have begun to characterize the similarities and differences between mice and human lungs after chronic cigarette smoke exposure. Using smoking chambers similar to those described in the past for other species, we found that mice tolerate at least two cigarettes daily for many months, resulting in many changes similar to human COPD (6). Unlike humans, mice are obligate nose-breathers; yet despite an intricate and extensive nasal sinus pathway, epithelial cells are olfactory in nature without extensive cilia and inefficiently filter tobacco smoke products. Mice have few submucosal glands that are located exclusively in the trachea. The airway is populated with epithelial cells and Clara cells but lacks true goblet cells. In C57BL/6 and A/J mice, ciliated epithelium extends throughout the airway with increasing proximal density. After two months of cigarette smoke, there is loss of ciliated epithelial cells, infiltration of immune and inflammatory cells (T cells, macrophages, neutrophils, and eosinophils), but no change in Clara cell numbers (Seoane, Lum, and Shapiro, unpublished observations). With prolonged cigarette smoke exposure (> 6 mo), small airways are occasionally obstructed by inflammatory cells and debris, and there are fewer alveolar attachments. Both of these changes in the small airways have been hypothesized to contribute to airflow obstruction in COPD. Mouse airways have much less extensive branching than humans and lack respiratory bronchioles. In murine alveolar spaces, we observe inflammatory cell recruitment and air-space enlargement in response to cigarette smoke, an effect similar to the response in humans. Further, there is increased alveolar duct area and enlarged alveolar spaces. Whether these pathologic changes are associated with abnormal pulmonary function or gas-exchange abnormalities awaits further study. Cigarette smoke–related changes appear to be strain-dependent, providing a unique opportunity to uncover COPD susceptibility genes.
Gene targeting or targeted mutagenesis by homologous recombination in embryonic stem cells has allowed investigators to generate strains of mice that lack individual proteins, providing specific loss of function models. Combination of gene targeting with the cigarette smoke–exposure model provides an opportunity to perform highly controlled experiments that differ with respect to expression of a single protein in mammals. Strains of mice deficient in individual candidate proteinases can be compared to determine their contribution to the development of emphysema in response to cigarette smoke. Macrophage elastase (MMP-12), nearly undetectable in normal macrophages, is expressed in human alveolar macrophages of cigarette smokers and in patients with emphysema but not in normal lung tissue. Exposure of macrophage elastase–deficient (MMP-12−/−) and wild-type (MMP-12+/+) littermates to chronic cigarette smoke exposure demonstrated that in contrast with MMP-12+/+ mice, MMP-12−/− did not develop emphysema (6). Surprisingly, MMP-12−/− mice also failed to recruit macrophages into their lungs in response to cigarette smoke. Gene targeting often leads to unexpected findings that lead to new, or in this case perhaps old, concepts. Our current working model demonstrates that cigarette smoke induces constitutive macrophages to produce MMP-12, which cleaves elastin, generating fragments chemotactic for monocytes. This positive feedback loop perpetuates macrophage accumulation and lung destruction. The concept that proteolytically generated elastin fragments mediate monocyte chemotaxis is not original. Independent studies by Senior and Mecham (7), as well as Hunninghake and Crystal (8), from the early 1980s demonstrated that elastase-generated elastin fragments were chemotactic for monocytes and fibroblasts. Gene targeting is merely reinforcing this as a major in vivo mechanism of macrophage accumulation in a chronic inflammatory condition. Although multiple proteinases from several cell types likely contribute with complex interactions to human emphysema, this study demonstrates that macrophage matrix metalloproteinases have the capacity to cause air-space enlargement in response to cigarette smoke; a concept previously considered a rumor.
Several spontaneous mutant mouse strains develop air-space enlargement (Table 1). These are usually developmental abnormalities rather than destruction of mature lung tissue characterizing emphysema. Tight skin (Tsk+/−) mice have a mutation in fibrillin-1, which is involved in elastic fiber assembly (9). These mice have abnormal air-space development and progressive alveolar enlargement with age. Pallid mice (pa/pa) develop mild emphysema late in life (10). The cause for this is unknown. While pallid mice, and in fact all C57BL/6 derivatives, have less α-1AT than other strains, antiproteinase activity should be sufficient to protect against spontaneous emphysema. Blotchy mice have enlarged air spaces believed to be a result of abnormal connective tissue or cross-linking (11). Recently, this mutation has been localized to abnormal RNA processing of the Menke gene on the X chromosome mottled locus (12). The relationship of this mutation with emphysema remains unknown. Beige mice (bg) have a defect in formation of primary granules (13, 14). It remains controversial whether they produce normal levels of serine proteinases and have the capacity to develop emphysema.
| Mutation* | Phenotype | Reference | ||||
|---|---|---|---|---|---|---|
| Natural genetic models | ||||||
| Tight skin (Tsk+/−) | fibrillin-1 | abnormal air-space development, progressive enlargement | (9) | |||
| Pallid (pa/pa) | unknown | develop mild emphysema late in life | ||||
| Blotchy (Blo) | Menke gene | enlarged air spaces (developmental?) | (12) | |||
| Transgenic mice | ||||||
| Collagenase | haptoglobin-Case | enlarged air spaces | (15) | |||
| IL-11 | CC-10-IL-11 | abnormal air-space development | (16) | |||
| PDGF-B | SP-C-PDGF-B | enlarged air spaces, fibrosis, inflammation | (18) | |||
| klotho | transgene disrupts klotho | progressive air-space enlargement after 2 wk | (2) | |||
| Gene-targeted mice | ||||||
| PDGF-A | null mutation | abnormal air-space development, lack myofibroblasts, decreased elastin | (19) | |||
| FGFR-3−/−XFGFR-4−/− | double null mutant | no alveolarization or secondary septation | (17) |
In addition to these naturally occurring mutations, several transgenic and gene-targeted mice develop enlarged air spaces (Table 1). Mice overexpressing collagenase (MMP-1) driven by the haptoglobin promoter somehow yielded lines of mice with transgene expression in the lung and resultant enlarged air spaces (15). Whether this is because of the destruction of collagen or interference with lung growth and development remains unclear. Lung-specific expression of interleukin (IL)-11 surprisingly led to enlarged air spaces, which was elegantly shown to be developmental in origin because inducible expression of IL-11 in adult mice displayed normal alveolar architecture (16). Lungs of mice deficient in both FGFR-3 and FGFR-4 (fibroblast growth factor receptors 3 and 4), but not deficient in either individually, are normal at birth but then fail to undergo alveogenesis and do not form secondary septae to delimit alveoli. Consequently, air spaces in the lung are expanded and no alveoli can be seen (17). Gene targeting of platelet-derived growth factor (PDGF)-B resulted in lack of myofibroblasts, with consequent decreased elastin synthesis and abnormal alveolar septation (18). Overexpression of PDGF-A led to air-space enlargement that could be secondary to fibrosis and airway tethering, although other explanations are possible (19). The transgene that is the topic of an article in this issue is the klotho mouse (2).
The klotho mouse, while fascinating in many respects, is also complicated and confusing. Klotho mice originated as the result of a failed transgenic experiment. The goal of the initial project was to generate transgenic mice overexpressing sodium channels in an attempt to develop a model of hypertension. Homozygous transgenic klotho founder mice that, although not hypertensive, displayed features of premature aging, including a short lifespan, infertility, arteriosclerosis, skin atrophy, osteoporosis, and “emphysema” (20). Further characterization demonstrated an insertion mutation of several copies of the transgene in the 5′-flanking region of what was termed the klotho gene, yielding a null mutation in klotho.1 Subsequent complementary DNA (cDNA) cloning demonstrated that klotho encoded a membrane protein with sequence homology to β–glucosidase enzymes. Specific functions of klotho and mechanisms of action remain unknown. It is believed that local expression of this transmembrane protein in the central nervous sysstem might ultimately lead to a diffusible factor responsible for aging.
It is not clear whether klotho mice have abnormal development or truly premature aging. For example, do their bones ever fully mineralize before becoming osteoporotic? And most relevant, do the lungs fully mature before becoming emphysematous? It is argued by Suga (2) that klotho mice have fully mature lungs before destruction. Yet alveolar enlargement begins after 2 wk of age, a period beyond alveogenesis but during lung growth. There is no data confirming “destruction” of air spaces, although it is a reasonable hypothesis. In fact, senile emphysema in humans is defined pathologically by enlarged alveolar ducts, as opposed to enlarged alveolar spaces. Of note, mice lack respiratory bronchioles and instead demonstrate enlargement of alveolar ducts in response to cigarette smoking. The klotho mouse phenotype prompts another interesting question: Is emphysema a prominent and consistent feature of the aging human lung? Clinically, at least, this does not appear to be a significant part of normal aging—again begging the question as to what the klotho mouse is modeling. These issues aside, the klotho mouse does have enlarged air spaces, and further study might unlock clues regarding lung development and repair.
The usefulness of animal models for COPD depends upon similar molecular mechanisms among species. With respect to lung structure, subtle differences in the alveolar space and gross differences in the airways—smoking mice simply aren't coughing up sputum—limit translation of findings among species. Nevertheless, mice appear to respond to cigarette smoke with low-grade chronic inflammation and lung destruction similar in humans. This makes study both difficult and informative. A role remains for simpler models such as elastase instillation, particularly to study mechanisms of lung repair, a critical area of investigation. More sophisticated physiologic and imaging analyses are needed to fully maximize the utility of animal models.
Originally, Gross instilled papain to induce granuloma formation, and Laurell and Eriksson only noticed in retrospect that patients deficient in α-1-AT had emphysema. Thus, the incidental klotho phenotype joins impressive company regarding insight into COPD that has come from studies originally intended for other purposes. Is it an accident that accidents have led to some of our greatest insights into the pathogenesis of emphysema? Could this be related to the fact that COPD is the illness least funded by the National Institutes of Health when adjusted for disease burden (21), or simply an unwillingness of investigators to study self-inflicted disease? Regardless of the cause, COPD research has been relatively stagnant, and new medical therapy has been lacking for decades.
This is particularly distressing given the devastating and increasing scope of COPD. In addition to the 16,000,000 Americans with COPD and its recent rise to the fourth leading cause of death in the United States, cigarette smoking is rampant worldwide, and cigarette-related deaths will overtake tuberculosis within a decade as the leading cause of death worldwide. In China alone, where 50% of long-term cigarette smokers develop COPD, there will be an estimated 1,500,000 deaths per year from COPD over the next half-century (22). Despite some tantalizing potential damage to the cigarette industry, there is no sign that cigarettes are going away. The good news is that from a scientific perspective, the time is right to understand and attack this disease. For those investigators however, who prefer to study other disease entities, please keep your eyes open for enlarged alveolar spaces. Maybe we can cure this disease without really trying.
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Abbreviations: α-1-antitrypsin, α-1AT; α-1AT deficiency, α-1A−/−; chronic obstructive pulmonary disease, COPD; interleukin, IL; collagenase, MMP-1; macrophage elastase, MMP-12; MMP-deficient mice, MMP-12−/−; wild-type mice, MMP-12+/+.
1 Note to young investigators: Don't try this at home. This difficult and risky task of tracing the genetic origins of this failed transgenic has resulted in a very interesting gene product. However, young investigators with limited time and resources are cautioned against chasing down mistakes, as they will most likely lead to career-ending dead ends.
