**5.1 Human COPD samples**

52 Chronic Obstructive Pulmonary Disease – Current Concepts and Practice

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

**4. Proteinase biology in cells relevant to COPD pathogenesis** 

degradation of these ECM proteins leads to the enlargement of lung airspaces.

*Lung inflammation and airspace enlargement* 

ciliary beat frequency of lung epithelial cells (48).

*Airway pathologies* 

The role of proteinases in COPD has been studied at a cellular level with *in vitro* studies.

The serine proteinases, NE, CG, PR3, and GRZ, can promote lung inflammation in COPD patients, through their direct action stimulating the release of pro-inflammatory mediators from airway epithelial cells and macrophages in vitro (35,36) and many proteinases also have an indirect action proteolytically cleaving mediators to alter their biologic activities. The metalloproteinases, MMPs-8 and -9, cleave and activate various chemokines *in vitro* (37,38). ADAM-17 and several MMPs shed and activate membrane-associated, latent proTNF- from macrophage surfaces (25,27,39). NE, MMP-12, and MMP-9 cleave elastin, and MMPs cleave AAT, generating fragments of these two molecules that are chemotactic for inflammatory cells (40,41). Serine, metallo-, and cysteine proteinases acting together can degrade elastin, interstitial collagens, and basement membrane proteins *in vitro* (7). The

The proteinases play a role in the characteristic airway pathologies of COPD, including increased mucus production, poor clearance of this mucus and resulting bacterial infections and further inflammation. NE, MMP-9, and ADAMs-10 and -17 increase epithelial cell expression of MUC5AC, a major mucin protein, by activating epithelial growth factor receptor (EGFR) through shedding of membranebound protransforming growth factor (TGF)-. The released soluble, active TGF-, activates the EGFR (42-44). The 3 major serine proteinases, NE, CG, and PR3 potently stimulate goblet cell degranulation (45). Tissue kallikrein is a serine proteinase expressed by inflammatory cells and submucosal glands, which also stimulates mucin synthesis in airway epithelium *in vitro* by shedding and activating pro-EGF, another EGFR ligand (46). NE damages epithelial cells (47) and inhibits

Plasmin, MMP-9, NE, and ADAMs may also induce sub-epithelial fibrosis in COPD airways, because they activate latent growth factors such as TGF-β (11,49,50) and insulinlike growth factors *in vitro* (51,52). These growth factors are known to induce fibroblasts to produce and secrete interstitial collagens. It remains unclear whether these proteinases

induce sub-epithelial fibrosis in the small airways of human COPD patients.

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 healthy subjects (53,55-65).

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.
