**6. Regulation of proteinases in the lung**

Proteinases are a significant factor in the pathogenesis of COPD, but do not act in isolation. They interact with other mediators and other pathways and are also regulated by inhibitors. Studies of the NE/ and MMP-12/ mice chronically exposed to cigarette smoke demonstrated interactions between these two classes of proteinases, with MMP-12 cleaving and inactivating AAT to increase NE-mediated lung injury, and NE cleaving and inactivating TIMP-1 to amplify MMP-12-mediated lung destruction (78). Proteinases also interact with reactive oxygen species (ROS), and ROS production is increased in the lungs of COPD patients. ROS are present in inhaled cigarette smoke itself, or are released by phagocytes activated by inhaled smoke. ROS are known to activate proMMPs *in vitro* and are thought to exacerbate lung inflammation and injury in COPD patients (92). Transgenic mice over-expressing the antioxidant enzyme Cu-Zn superoxide dismutase in the lung are protected from developing chronic lung inflammation, increased lung MMP levels, and emphysema in response to intratracheal instillation of porcine pancreatic elastase, or chronic exposure to cigarette smoke (93). However, mice deficient in a phagocyte-specific component of the NADPH oxidase, which generates superoxide anions (O2- ), develop greater airspace enlargement in response to cigarette smoke than WT mice (94). This is due to ROS-mediated inactivation of MMPs via oxidative inactivation of residues in the catalytic domain of MMPs (95). Thus, phagocyte-derived O2- (and ROS derived from O2-) in COPD lungs may constrain rather than promote phagocyte MMP-mediated lung injury (94,96). It is noteworthy that clinical trials have failed to demonstrate protective effects of antioxidant supplementation in COPD patients, and this could be linked, in part, to antioxidants inducing reductions in ROS-mediated inactivation of MMPs (97).

#### **6.1 Inhibitors of proteinases**

Proteinase inhibitors are present in the extracellular matrix. To maintain their action, proteinases need to circumvent these inhibitors through inactivation of the proteinase inhibitor, evading them and / or overwhelming them.

#### **6.2 Inactivation of proteinase inhibitors**

Serpins can be cleaved and inactivated by MMPs (98-102), NE (103,104), cathepsin B (105), and bacterial proteinases (106). Serine proteinases cleave and inactivate TIMPs (107).

Diverse Activities for Proteinases in the

individual cells (quantum proteolysis).

*Brisk recruitment of inflammatory cells in the lung* 

*Quantum proteolysis and PiZZ AAT deficiency* 

are also chemotactic for PMN (136,137).

**mediated lung pathologies in COPD** 

**7.1 Proteinase inhibition** 

Pathogenesis of Chronic Obstructive Pulmonary Disease 57

large numbers of inflammatory cells, or when high concentrations are released from

COPD exacerbations are characterized by an influx of inflammatory cells into the airways. These cells release active forms of NE, MMP-8, and MMP-9 (58,62,129,130). Macrophage clearance of the PMN recruited into the lung under normal circumstances would occur but in the case of the COPD lung this is hampered by a number of mechanisms. First, cigarette smoke impairs expression of recognition molecules for apoptotic PMN on the macrophage surface (131). Second, NE cleaves recognition molecules for apoptotic PMN from the macrophage surface (132). Third, when PMN ingest *Hemophilus influenzae,* which frequently colonizes the respiratory tract of COPD patients, PMN necrosis is rapidly induced (133).

NE is present at millimolar concentrations in each azurophil granule of PMN, which is more than 100-fold higher than the concentration of AAT, its inhibitor, in plasma (134). The release of an azurophil granule into the extracellular space is thus accompanied by a transient burst of proteolytic activity as it greatly outnumbers the proteinase inhibitors. This activity fades as the granule contents diffuse, and the proteinase-inhibitor ratio falls below 1:1 (134). In patients with an inherited deficiency of AAT, the proteinase activity lasts longer, leading to more destruction of the lung. Quantum bursts of NE-mediated proteolytic activity associated with PMN migrating on ECM proteins are 10-fold larger in area and 4 fold longer in duration when PMN are bathed in serum from PiZZ patients compared to serum from healthy PiMM subjects (135), due to defective confinement of PMN-derived NEmediated ECM degradation. The PiZ AAT mutant proteins polymers formed in this disease

**7. Potential strengths and limitations of proteinase inhibitors and antiinflammatory drugs as new therapeutic strategies to limit proteinase-**

Perhaps the most obvious role for intervention in this setting is to replace AAT in patients with COPD who have known severe, inherited AAT deficiency (AATD). Although we do not have conclusive randomized controlled trials, human clinical research has shown that AAT augmentation reduced exacerbation frequency and slows the rate of lung function decline in these patients (138). More recent work has attempted augmentation of AAT through gene therapy. This involves administration of recombinant adeno-associated virus (rAAV) vectors expressing human AAT (rAAV1-CB-hAAT) to patients with AATD (139). These studies are currently in phase 2 clinical trials and have shown increased expression of normal (PiM) AAT in serum occurs safely in patients for up to 90 days. Further optimization of the vector is likely to be required to generate sustained therapeutic AAT plasma levels. The concept of augmentation of AAT in COPD, outside the setting of AATD, is less clear.

Secretory leukocyte peptidase inhibitor (SLPI) and elafin are naturally occurring antiproteinases with anti-NE activity whose roles in COPD are not fully eludicated but may have potential as future treatment options (140). A number of synthetic low molecular

Proteolytic inactivation of AAT and TIMP-1 by MMP-12 and NE occurs in the cigarette smoke exposure model of emphysema in mice (78). ROS present in cigarette smoke or released by leukocytes activated by smoke, inactivate α2-M, and AAT, and SLPI in vitro by converting the methionine at the active sites of these inhibitors to methionine sulfoxide. This reduces their capacity to inhibit serine proteinases (108-111). It is not clear if oxidative inactivation of proteinase inhibitors occurs in COPD patients. Some studies have detected oxidized AAT in lung samples from COPD patients but others have not (112-114). Also, ROS can inactivate proteinases as outlined above. It is difficult to know if previous work analyzing the oxidation state of proteinase inhibitors in lung samples from COPD patients actually includes events in cellular microenvironments. Adding to the complexity of studying this process is the fact that ROS are short-lived molecules and are active only at short distances from the cells generating them before they are muted by antioxidants.

### **6.2.1 Evasion of inhibitors**

In another effort to preserve their function, proteinases can evade inhibitors by binding tightly to substrates, being released into sequestered microenvironments, or binding to cell surfaces.

#### *Tight binding of proteinases to substrates*

NE binds very stably to elastin in an active form, and AAT and SLPI have reduced activity against elastin-bound NE compared to soluble NE (54,115,116). In the lungs of humans with emphysema, NE is bound to interstitial elastin (53) and this lung elastin-bound NE likely retains catalytic activity and takes a major role in the destruction of elastin fibers in pulmonary emphysema (Fig. 4). MMPs-1, -2, and -9 bind to various ECM proteins, which may increase the retention, stability, and bioactivity of proteinases in the lung and aid their roles in extracellular proteolysis (117,118).

#### *Sequestered microenvironments*

Inflammatory cells can, via integrin-mediated adhesion to matrix or to cells, form small pockets of microenvironment. Large inhibitors such as AAT (119) and α2-M (120) cannot enter these sealed pockets (Fig. 4).

#### *Membrane binding of proteinases*

MT-MMP and ADAMs are integral membrane proteinases, and some members of these families are resistant to inhibition by physiologic inhibitors. ADAM-17, for example, is resistant to inhibition by TIMPs-1 and -2 but not TIMP-3 (31), and MT1-MMP is resistant to inhibition by TIMP-1 but not TIMP-2 (121). NE, CG, PR3, MMPs-8 and -9 (which lack transmembrane domains or glycosylphosphatidyl-inositol anchors) are also expressed on the surface of activated PMN (122-127). These surface-bound proteinases degrade lung ECM proteins and proteinase inhibitors and induce goblet cell degranulation (122,126-128). The membrane-bound element of these proteinases confers a resistance to their inhibitors when compared to the soluble variety (122-124,126,127).

#### **6.2.2 Overwhelming of inhibitors**

A more obvious way to overcome the inhibitors is for the proteinases to overwhelm them with sheer numbers. This can happen with release of massive quantities of enzymes from

Proteolytic inactivation of AAT and TIMP-1 by MMP-12 and NE occurs in the cigarette smoke exposure model of emphysema in mice (78). ROS present in cigarette smoke or released by leukocytes activated by smoke, inactivate α2-M, and AAT, and SLPI in vitro by converting the methionine at the active sites of these inhibitors to methionine sulfoxide. This reduces their capacity to inhibit serine proteinases (108-111). It is not clear if oxidative inactivation of proteinase inhibitors occurs in COPD patients. Some studies have detected oxidized AAT in lung samples from COPD patients but others have not (112-114). Also, ROS can inactivate proteinases as outlined above. It is difficult to know if previous work analyzing the oxidation state of proteinase inhibitors in lung samples from COPD patients actually includes events in cellular microenvironments. Adding to the complexity of studying this process is the fact that ROS are short-lived molecules and are active only at short distances from the cells generating them before they are muted by antioxidants.

In another effort to preserve their function, proteinases can evade inhibitors by binding tightly to substrates, being released into sequestered microenvironments, or binding to cell

NE binds very stably to elastin in an active form, and AAT and SLPI have reduced activity against elastin-bound NE compared to soluble NE (54,115,116). In the lungs of humans with emphysema, NE is bound to interstitial elastin (53) and this lung elastin-bound NE likely retains catalytic activity and takes a major role in the destruction of elastin fibers in pulmonary emphysema (Fig. 4). MMPs-1, -2, and -9 bind to various ECM proteins, which may increase the retention, stability, and bioactivity of proteinases in the lung and aid their

Inflammatory cells can, via integrin-mediated adhesion to matrix or to cells, form small pockets of microenvironment. Large inhibitors such as AAT (119) and α2-M (120) cannot

MT-MMP and ADAMs are integral membrane proteinases, and some members of these families are resistant to inhibition by physiologic inhibitors. ADAM-17, for example, is resistant to inhibition by TIMPs-1 and -2 but not TIMP-3 (31), and MT1-MMP is resistant to inhibition by TIMP-1 but not TIMP-2 (121). NE, CG, PR3, MMPs-8 and -9 (which lack transmembrane domains or glycosylphosphatidyl-inositol anchors) are also expressed on the surface of activated PMN (122-127). These surface-bound proteinases degrade lung ECM proteins and proteinase inhibitors and induce goblet cell degranulation (122,126-128). The membrane-bound element of these proteinases confers a resistance to their inhibitors when

A more obvious way to overcome the inhibitors is for the proteinases to overwhelm them with sheer numbers. This can happen with release of massive quantities of enzymes from

**6.2.1 Evasion of inhibitors** 

*Tight binding of proteinases to substrates* 

roles in extracellular proteolysis (117,118).

compared to the soluble variety (122-124,126,127).

*Sequestered microenvironments* 

enter these sealed pockets (Fig. 4). *Membrane binding of proteinases* 

**6.2.2 Overwhelming of inhibitors** 

surfaces.

large numbers of inflammatory cells, or when high concentrations are released from individual cells (quantum proteolysis).

#### *Brisk recruitment of inflammatory cells in the lung*

COPD exacerbations are characterized by an influx of inflammatory cells into the airways. These cells release active forms of NE, MMP-8, and MMP-9 (58,62,129,130). Macrophage clearance of the PMN recruited into the lung under normal circumstances would occur but in the case of the COPD lung this is hampered by a number of mechanisms. First, cigarette smoke impairs expression of recognition molecules for apoptotic PMN on the macrophage surface (131). Second, NE cleaves recognition molecules for apoptotic PMN from the macrophage surface (132). Third, when PMN ingest *Hemophilus influenzae,* which frequently colonizes the respiratory tract of COPD patients, PMN necrosis is rapidly induced (133).

#### *Quantum proteolysis and PiZZ AAT deficiency*

NE is present at millimolar concentrations in each azurophil granule of PMN, which is more than 100-fold higher than the concentration of AAT, its inhibitor, in plasma (134). The release of an azurophil granule into the extracellular space is thus accompanied by a transient burst of proteolytic activity as it greatly outnumbers the proteinase inhibitors. This activity fades as the granule contents diffuse, and the proteinase-inhibitor ratio falls below 1:1 (134). In patients with an inherited deficiency of AAT, the proteinase activity lasts longer, leading to more destruction of the lung. Quantum bursts of NE-mediated proteolytic activity associated with PMN migrating on ECM proteins are 10-fold larger in area and 4 fold longer in duration when PMN are bathed in serum from PiZZ patients compared to serum from healthy PiMM subjects (135), due to defective confinement of PMN-derived NEmediated ECM degradation. The PiZ AAT mutant proteins polymers formed in this disease are also chemotactic for PMN (136,137).
