PERSPECTIVE
Neutrophil Elastase
Path Clearer, Pathogen Killer, or Just Pathologic?

Steven D. Shapiro

Division of Pulmonary and Critical Care Medicine, Department of Medicine, Brigham and Women's Hospital, Harvard University Medical School, Boston Massachusetts

Serine proteinases have diverged evolutionarily from a single gene product undergoing duplication and mutations yielding enzymes with diverse biologic functions, including digestive enzymes of exocrine glands, clotting factors, and leukocyte granule-associated proteinase such as neutrophil elastase. Neutrophil elastase (NE) is a 30-kD glycoprotein chymotrypsin-like serine proteinase with potent catalytic activity dictated by a catalytic triad that consists of His, Asp, and Ser residues that form a charge relay system (1). NE, along with cathepsin G (CG), and proteinase 3 (PR3), are formed in a lineage-restricted and developmentally specific manner during the development of myeloid cells, and stored in granules as active packaged proteins. Despite extensive knowledge of NE, the enzyme, we still struggle to understand its biologic function(s). Whereas the role of other serine proteinases as digestive enzymes and clotting factors is clear, most of our knowledge regarding NE revolves around its capacity to cause tissue destruction. Indeed, NE appears to cause more trouble than it is worth. This has been the basis for development of inhibitors, including the novel and potent EPI-HNE-4, described by Delacourt and coworkers in this issue (2).

NE has been estimated to be present at high concentrations (5 mM) in neutrophil azurophil granules. It is hard to believe that this destructive enzyme would be tolerated in such large amounts if it did not serve important biologic functions. Moreover, the fact that ₁-antitrypsin (₁-AT) is one of the most abundant extracellular proteins, and a prominent part of the acute phase response, suggests that NE is not meant to roam the extracellular space. When optimally primed by biologically relevant agonists, neutrophils release less than 2% of their total NE into the extracellular space, but they are able to translocate by "quantum bursts" of granules as much as 12% of their total NE to the cell membrane where the enzyme is active and resistant to inhibition (3). NE could be released from the cell during "frustrated phagocytosis" and following cell death if not taken up by macrophages. It is under these circumstances that NE is probably most destructive.

	Pathogen Killer

Given its high intracellular concentrations within the neutrophil, it is logical to postulate a role for NE as an antimicrobial agent. Indeed, Janoff and coworkers provided in vitro data that NE exhibits antibacterial activity many years ago (4), although NE had not been generally considered a significant antimicrobial agent. Gene targeting of NE has directly supported important roles of NE in host defense. Mice deficient in NE have impaired survival to Gram-negative infections (5). NE is required for efficient intracellular killing of Gram-negative, but not Gram-positive, bacteria. NE acts via catalytic proteolysis of specific Gram-negative outer wall proteins (Omps) (6). A second group independently targeted NE and their NE^/ mice displayed impaired killing of bacteria and fungi (7). NE might have its greatest effect in the GI tract, where it has been recently shown that NE degrades toxins of Shigella, Salmonella, and Yersinia 1,000 times more effectively than other bacterial proteins, and prevents escape of Shigella from neutrophil phagosomes (8). It should be noted that NE^/ mice do not appear to be immunocompromised; that is, they are not at increased risk of spontaneous infection. The importance of NE-mediated bactericidal activity is unmasked upon overwhelming Gram-negative bacterial infection.

	Path Clearer

Cell surface-bound NE would be ideally suited to provide focal proteolysis and "clear a path" for migrating neutrophils. Yet there has been little evidence to support this notion. In the article by Delacourt and coworkers, they were able to inhibit human neutrophil migration through a synthetic basement membrane (Matrigel) by their NE inhibitor. Previously, this group showed that matrix metalloproteinase (MMP)-9 inhibition also prevented neutrophil migration through Matrigel (9). They surmise that NE activates pro-MMP-9, and MMP-9 mediates matrix degradation "creating a path" for the neutrophil to migrate. These findings are in contrast to a classic manuscript by Weiss and colleagues (10) in which they failed to inhibit human neutrophil migration through endothelial basement membranes with any class of proteinase inhibitor. Recently, O'Connor reported in this journal that using newer small molecular weight synthetic MMP and serine proteinase inhibitors, they still could not inhibit neutrophil migration ex vivo (11). Similarly, neutrophils from MMP-9-deficient gene targeted mice migrate normally through tissue barriers in vitro and in vivo in both systemic and pulmonary circulation (12). NE-deficient neutrophils also migrate normally through Matrigel; however, NE does appear to be required for neutrophil migration into the lung in response to some, but not all stimuli (Shapiro, unpublished results). These findings are consistent with the need of NE to detach neutrophil CD11b/18 from vascular intercellular adhesion molecule (ICAM).

It is notable that mechanisms of neutrophil transvascular migration in the systemic circulation differ from the pulmonary circulation (13). In the systemic (and bronchial) circulation, neutrophils egress through postcapillary venules via several steps, including L-selectin-mediated rolling, firm attachment via ICAM, and migration between endothelial cells directed by P-selectin. In the pulmonary capillary, the activated neutrophil becomes "stiff" and physical constraints limit transit through the capillary independent of L-selectin. Neutrophils then become deformable, "slithering" through cellular and matrix barriers into the lung parenchyma, perhaps independent of proteinases. Is it possible that EPI-HNE-4 interferes with the ability of the neutrophil to change shape? Further studies will be required to explain these interesting and seemingly contradictory phenomena.

	Pathologic

There is little argument that uninhibited NE exposed to tissues will cause damage. Its destructive role was solidified almost 40 years ago when Laurell and Eriksson reported an association of chronic airflow obstruction and emphysema with deficiency of serum ₁-AT (14), the endogenous inhibitor of NE. Moreover, instillation of NE in lungs of experimental animals resulted in emphysema. We now appreciate that other proteinases might contribute to emphysema, but a significant role for NE remains likely. In addition to its matrix-destructive effects, NE is a potent secretagogue and induces muc 5A expression contributing to excess mucus production in chronic obstructive pulmonary disease (COPD).

NE is also associated with other acute and chronic lung diseases. As described in this issue by Delacourt and coworkers (2), NE is present in CF airways and thought to have multiple adverse effects in CF, including airway tissue remodeling preventing bacterial clearance, immunoglobulin degradation, and stimulating IL-8 production contributing to uncontrolled inflammation. NE and elastin fragments have also been found in ARDS, and NE inhibitors have been shown to decrease inflammation and lung edema in animal models of acute inflammation (15).

	NE Inhibition

Given its destructive capacity, NE would be a desirable target for a variety of lung diseases. Despite its beneficial roles, NE can be safely inhibited, extracellular inhibition being safest and intracellular inhibition potentially most effective. Limited human trials have not reported adverse effects even in patients with CF who are colonized by bacteria. It is possible that NE is less important in bacterial killing in human than mouse neutrophils. More likely, the need for NE may only be unmasked by massive infection when the neutrophil needs its full arsenal of weapons. This should raise caution if patients undergoing NE therapy become septic. Nevertheless, given the destructive effects of NE, its inhibition remains an attractive concept for several disease states.

Given a wealth of effective NE inhibitors developed over the years, including EPI-HNE-4 introduced by Delacourt and colleagues in this issue of the journal, why then haven't they advanced as therapeutic targets? It is often assumed that they must have failed, when in fact they really have not been fully tested. Most activity has been in the area of COPD, where the common scenario is that NE is identified as a target, effective inhibitors are developed, and early phase trials for safe and effective NE inhibition. Then reality hits. COPD clinical trials will be expensive, requiring many patients and long-term studies. To further complicate the situation, we still lack effective biomarkers to provide interim confidence that the drug is having its intended effect. Thus, the combination of expense and risk have thus far prevented development of this class of inhibitors; in fact, these issues have limited all COPD pharmacotherapy to date. Hopefully, as we enter the era of improved imaging and proteomics, biomarkers will become available and NE inhibitors and other forms of COPD therapy will finally be tested. It is likely that NE inhibitors will be first developed for acute inflammatory lung diseases such as adult respiratory distress syndrome. Although one can empathize with the difficult task of drug development, it would be pathetic if we never develop the full potential of NE inhibition.

	Footnotes

Address correspondence to: Steven D. Shapiro, M.D., Parker B. Francis Professor of Medicine, Brigham and Women's Hospital, Harvard Medical School, 75 Francis Street, Boston, MA 02115. E-mail: sshapiro{at}rics.bwh.harvard.edu

(Received in original form January 29, 2002).

	References

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