**3. Evidence for activities for proteinases in COPD**

The proteinase/anti-proteinase hypothesis for the pathogenesis of COPD is not a new concept. It dates back to experimental work done over 50 years ago. The basis of the concept was 2 key observations. The first came from the keen observations by Laurell and Eriksson who noted that deficiency of AAT was associated with early onset, severe panlobular emphysema (33). AAT has since been shown to be the major inhibitor of NE in the lower respiratory tract. The second observation was made when instillation of papain (an enzyme with elastase activity) into rat lungs was shown to cause progressive airspace enlargement (34). Over the years, other elastolytic proteinases have been shown to cause airspace

Diverse Activities for Proteinases in the

**pathologies in COPD patients** 

models of COPD.

**5.1 Human COPD samples** 

healthy subjects (53,55-65).

**5.2 Animal models of COPD** 

*Acute cigarette smoke exposure models* 

a reduced risk of COPD in adult smokers (72).

COPD.

Pathogenesis of Chronic Obstructive Pulmonary Disease 53

Most of the evidence for the mechanisms by which the proteinases act in the disease process of COPD comes from studies of clinical samples from human COPD patients and animal

Following on from the initial discovery that lack of inhibition of NE in patients with AAT deficiency was associated with emphysema, studies from Damiano et al further supported crucial activities for NE in pulmonary emphysema (53). They showed that the amount of NE bound to lung elastin is strongly correlated with the degree of emphysematous change and additional studies demonstrated stable binding of active forms of NE to elastin *in vitro* (54). Since then, additional studies have confirmed increased levels of NE in lung samples from COPD patients and demonstrated elevated levels of CG, PR3, uPA, and MMPs -1, -2, -8, -9, and -14 in various lung samples from smokers and COPD patients when compared to

Inflammatory cells are the main source of these proteinases in COPD but production of proteinases by lung structural cells and immune cells has also been demonstrated. For example, cigarette smoke increases MMP production by lung epithelial cells (64), and fibroblasts (66). T lymphocytes from blood and BAL samples from COPD patients have increased levels of GRZ and perforin compared to samples from asymptomatic smokers and nonsmokers (67). Elevated levels of GRZ B in BAL samples from COPD patients show a correlation with bronchial epithelial cell apoptosis, suggesting that GRZ B promotes epithelial cell death in the lung and contributes to airspace enlargement in COPD patients.

Animal models of COPD provide the strongest evidence for the roles of proteinases in

Exposing mice to smoke for up to 30 days leads to an influx of PMN and macrophages to the lung (68). This is due to direct effects of inhaled smoke on lung capillaries, leading to leakage of thrombin and plasmin into the alveolar space (69,70). These proteinases cleave and activate PAR-1 on macrophages, leading to an increased synthesis of MMP-12 by macrophages (13,14). MMP-12 is responsible for shedding pro-TNF-α from activated macrophages, likely leading to an increase in E-selectin expression on endothelial cells (39). This facilitates transendothelial migration of PMNs. The presence of these increased PMNs and macrophages, releasing serine proteinases, increases lung collagen and elastin breakdown. Delivering human AAT to mice acutely exposed to cigarette smoke prevents PMN influx and ECM destruction. This is probably due to AAT inhibiting both PMN serine proteinase-mediated ECM destruction and thrombin- or plasmin-induced increases in macrophage MMP-12 production (14,71). Further evidence for the role of MMP-12 comes from a study showing that the minor allele of a single-nucleotide polymorphism (SNP) in MMP-12, is associated with a positive effect on lung function in adults who smoke and also

**5. Mechanisms by which proteinases contribute to individual lung** 

enlargement when instilled into the lungs of animal models. The concept proposed that the imbalance between proteinases (especially elastases) and their inhibitors lead to pulmonary emphysema. Emphysema, however, does not account for all COPD patients and whereas AAT deficiency is a cause of COPD, AAT deficiency only accounts for approximately 2% of COPD. Other factors have now been implicated in airspace enlargement in COPD, including other classes of proteinases (MMPs and cysteine proteinases), oxidative stress, and apoptosis of lung structural cells. COPD is a clinically and pathologically heterogeneous disease and includes chronic inflammation in the alveolar space, airways, and lung interstitium; mucus hypersecretion; and subepithelial fibrosis in the small airways. Although the proteinase/ antiproteinase concept does not account for all of the complex pathologies that make up COPD it certainly has far-reaching effects, many of which have been investigated in *in vitro* studies, and studies of human samples from COPD patients and animal models of COPD
