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

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

#### *Lung inflammation and airspace enlargement*

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

#### *Airway pathologies*

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 ciliary beat frequency of lung epithelial cells (48).

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.
