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"Knock-out" Mouse

Down But Not Out

Queen Elizabeth Hospital Birmingham, England

Emphysema is one component of chronic obstructive pulmonary disease (COPD) and at present there is no specific treatment other than smoking cessation. Understanding the pathogenesis of the process is central to the development of appropriate therapy.

Proteinases are thought to be the mediators of the tissue damage and their activity can be inhibited by both natural and synthetic inhibitors. Before such approaches can be tested and adopted in man, however, it is essential that the key proteinases responsible for the development of emphysema are identified.

In humans, ₁-antitrypsin deficiency is the only recognized risk factor and neutrophil enzymes inhibited by this protein, such as neutrophil elastase, proteinase 3, and cathepsin G, all generate features of COPD in animal models (1, 2). For these reasons, neutrophil elastase in particular has always been a key target in the development of new therapeutic strategies. In 1997, Hautamaki and colleagues (3) demonstrated that knocking out a different proteinase (mouse metalloelastase or matrix metalloproteinase [MMP] 12) prevented the subsequent development of emphysema in the smoking mouse. Because this enzyme has different properties and, hence, inhibitor profile to neutrophil elastase, the whole strategic approach to emphysema was turned on its head. In the current issue of AJRCCM (pp. 199–207), however, the article by Churg and coworkers (4) brings the story full circle and once again implicates serine proteinases in the development of emphysema. Of importance, the authors suggest that augmentation of ₁-antitrypsin, even in patients without genetic deficiency, may play a role in the management of human emphysema.

In particular, this article brings together several aspects of the emphysema generating process into a unified concept where a circle of inflammatory processes can be broken by the introduction of an external mediator.

The authors previously confirmed that smoking leads to development of emphysema in the mouse associated with neutrophilic inflammation (5) related to tumor necrosis factor- expression mediated by MMP12 (6) and lung connective tissue breakdown (5, 7). Synthetic neutrophil elastase inhibitors prevent the development of emphysema after smoking in the guinea pig (8), but studies investigating the role of human ₁-antitrypsin have been hampered because it is immunogenic to nonhuman species.

To address this problem, the authors have developed transgenic mice that express low levels of human ₁-antitrypsin. This essentially induces tolerance in mice to the human protein and enables repeated treatments to be given with prolastin. This product, however, also contains other human proteins (especially albumin), which should be immunogenic to the mouse. Nevertheless, the strategy of repeated dosing does not appear to be harmful.

With this as a background, the authors compared the airway inflammation, tumor necrosis factor- expression, connective tissue breakdown, and development of emphysema in control mice and in mice treated with prolastin injected intraperitoneally. The treatment normalized neutrophil influx, plasma tumor necrosis factor-, and lung parenchymal tissue degradation products, and prevented the development of emphysema.

Superficially, these findings reestablish a key role for neutrophil elastase in the pathogenesis of emphysema by breaking the cycle of events (see figure in online supplement).

However, there are several features about the current article that I find intriguing and require further comment and thought. First, if the series of events implicated by the authors is correct, interrupting the biological effects of elastase should prevent the tissue damage, release of chemotactic degradation products (9), and subsequent neutrophil recruitment; it should not, however, alter the production and release of tumor necrosis factor- as a result of MMP12 released by the macrophage.

Second (and perhaps more importantly), ₁-antitrypsin, which has had its active site methionine oxidized (thereby impairing its function), has the same effect as native ₁-antitrypsin. The authors have shown that this is not due to reactivation of the ₁-antitrypsin in the blood but they failed to confirm that this ₁-antitrypsin does not inhibit mouse neutrophil elastase. This point is important because oxidized ₁-antitrypsin is not inactive against human neutrophil elastase, but it does have a 2,000-fold reduction in its association rate-constant for neutrophil elastase (10). It is unknown how oxidation affects the ability of ₁-antitrypsin to inhibit mouse neutrophil elastase. This begs the question "What does oxidized AAT do?" First, it could have a non-proteinase effect; perhaps it binds active components in cigarette smoke such as oxidants, reducing them by alternative methionine residues on the protein. This effect could block MMP12 release and thereafter tumor necrosis factor- production. Second, injecting the oxidized ₁-antitrypsin (which is proinflammatory) into the peritoneal cavity could lead to sequestration of the neutrophils, therefore preventing them from being sequestered in the lung. Unfortunately, information on the abdominal pathology is missing in the current report.

Finally, if oxidized ₁-antitrypsin is proinflammatory it may increase the production of mouse proteinase inhibitors within the lung, thereby dampening the subsequent inflammatory response to cigarette smoke. Again, information on this possibility is lacking.

The animals were treated 24 hours before the first exposure to cigarette smoke. It would be of importance to demonstrate efficacy once the inflammatory cycles have been established. This is likely to be how such treatments would be used in the human condition because patients would have to have established disease before symptoms that lead to medical advice and treatment.

The current article by Churg and colleagues poses intriguing questions that need further exploration and clarification. In particular, the lack of development of emphysema in the MMP knock-out mouse needs to be reconsidered to determine the real role of MMP12 in the proinflammatory cascade that leads to the development of emphysema. Questions about the true role of ₁-antitrypsin as a protein and an elastase inhibitor need to be reconsidered and these studies should lead to exploration of the more detailed aspect of the pathogenic pathway.

Finally, it is of interest that these mice develop emphysema even in the presence of their own ₁-antitrypsin and that augmentation abrogates the process. Again, this observation has major implications for human emphysema not related to ₁-antitrypsin deficiency because the data suggests that augmentation of proteinase inhibitors would still have a role in such subjects.

Acknowledgments

The author thanks R. Lewis for typing and A. Munro for helping formulate ideas.

REFERENCES

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