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You May Say That I Ain't Free—But It Don't Worry Me^*

Laboratorio di Biochimica e Genetica Clinica di Malattie dell'Apparato Respiratorio IRCCS Policlinico San Matteo Università degli Studi di Pavia Pavia, Italy

Concepts concerning the pathogenesis of chronic obstructive pulmonary disease are rapidly evolving, and lessons learned from genetically manipulated animals indicate that airspace enlargement is a complex phenomenon deriving from a number of mechanisms (1). Nevertheless, the proteinase–antiproteinase hypothesis still dominates nearly 40 years after its original postulation. Although other characters such as collagens, matrix metalloproteinases, and their inhibitors have reached the stage in the last decade (2), neutrophil elastase still shares the limelight, along with ₁-antitrypsin (₁-AT).

Evolving concepts about the pathogenesis of chronic obstructive pulmonary disease have not spared neutrophil elastase. We now look at this proteolytic enzyme not just as the effector of uncontrolled interstitial elastin breakdown, but also as an agent able to act, at least in vitro, on a variety of substrates, including other extracellular matrix proteins, surface cell receptors, ligands, cytokines, immunoglobulins, and proteinase inhibitors (3), thus orchestrating many inflammatory mechanisms: in a proinflammatory fashion in some cases and in an downregulatory fashion in others. The concept of neutrophil elastase having broad substrate specificity is particularly relevant to the pathogenesis of chronic bronchitis (4), because consequences of the proteinase–antiproteinase imbalance on elastin breakdown in the airways play little, if any, role.

Much effort has been spent in understanding the mechanisms shifting neutrophil elastase and ₁-AT from their physiologic balance. Most studies have dealt with the antiproteinase side of this balance, and so we now know that the decrease in ₁-AT may result from a variety of conditions, including inherited deficiency, conformational aberration, oxidation, and proteolytic cleavage. Little attention, however, has been paid to the mechanisms of the proteinase counterpart, beyond the generic concept that neutrophil elastase is discharged by recruited and triggered neutrophils. There is in vitro evidence, however, that several factors may modulate neutrophil elastase activity. Interestingly, polyanions existing in lung secretions, such as DNA and glycosaminoglycans, bind neutrophil elastase and decrease the rate of its inhibition by ₁-AT (5–7).

In this issue of AJRCCM (pp. 192–198), Chan and colleagues (8) provide evidence that these kinds of supramolecular complexes are present in vivo in lung fluids, resulting in modulated neutrophil elastase activity. As a model, they investigated specimens obtained from patients affected by idiopathic disseminated bronchiectasis, but their results are potentially transferable to individuals with commoner causes of chronic airway inflammation. By subjecting sputum sol phases to casein zymography, the authors demonstrated proteolytic activity in a zone at a molecular mass (250–90 kD) higher than that displayed by free neutrophil elastase (30 kD). Western blot analysis revealed that the complex was formed by neutrophil elastase and syndecan-1, a transmembrane heparan sulfate proteoglycan expressed on bronchial epithelial cell surfaces and shed in inflammatory fluids. No caseinolytic activity was found in the zone where "free" neutrophil elastase (30 kD) is expected to act. Chan and colleagues (8) performed a number of additional experiments to confirm the validity of their findings. Interestingly, Western blots also demonstrated that the two major, naturally occurring antiproteinases, ₁-AT and secretory leukoproteinase inhibitor, are detectable in the complex; nevertheless, the free activity of neutrophil elastase suggests that they are ineffective in preventing proteolysis.

A large body of literature on free neutrophil elastase activity in bronchial secretions has accumulated over the last 25 years (9), and much attention has been focused on mechanisms of impairment of antiproteinase activity. An important point addressed in the article by Chan and coworkers (8) is that, when exogenous ₁-AT is added to the sputum sol, the inhibitor:enzyme binding stoichiometry shifts from 1:1 (the expected stoichiometry, when purified and active site titrated ₁-AT and neutrophil elastase are mixed in vitro [10]) to about 10:1, without reaching complete inhibition. It is therefore likely that proteoglycan-bound neutrophil elastase is rendered less accessible to the ₁-AT inhibitory site. Another interesting finding is that the complex seems to enhance the inhibitory activity of exogenous secretory leukoproteinase inhibitor, thus suggesting that the proteoglycan-induced activation of secretory leukoproteinase inhibitor, because of conformational changes in the inhibitor (11), may also take place in real samples.

So, what is "free" in inflamed bronchial secretions is not neutrophil elastase, but its activity, and binding to proteoglycans modulates the inhibition by antiproteinases.

The article by Chan and colleagues opens up speculations related to therapeutic approaches to proteinase–antiproteinase imbalance. Supplementation therapy with intravenous ₁-AT is currently given to patients with ₁-AT deficiency. Interestingly, this therapy failed to reduce excretion of elastin breakdown products (12), the free neutrophil elastase activity being partially unaffected, despite the significant increase in ₁-AT levels in sputum sols, with an ₁-AT:neutrophil elastase ratio on the order of micromolar:nanomolar (13). Binding of neutrophil elastase to proteoglycans, as shown by Chan and colleagues, is a potential explanation for these findings, and it should be taken into account for future studies, such those planned with inhaled ₁-AT.

Many steps in the proteinase–antiproteinase hypothesis have been postulated on the basis of results obtained in vitro with purified components. But real samples are microenvironments in which single components may interact, with unexpected results. The findings of Chan and colleagues, although achieved by relatively simple biochemical techniques, are an example of an unexpected interaction, simply because one of the components (i.e., proteoglycans) was not previously investigated in vivo under this aspect. Many other supramolecular interactions might take place in vivo (14, 15). Proteomics is a discipline that can rapidly give us a better understanding of the protein composition of real samples: just as functional genomics is the step beyond genomics, so functional proteomics would provide us with a comprehensive view of protein interactions and their functional consequences.

FOOTNOTES

^* "It Don't Worry Me," song composed by Keith Carradine for Nashville, a film produced and directed by Robert Altman. Paramount Pictures, 1975.

Conflict of Interest Statement: M.L. received $10,000 for serving as a consultant to Pharmacia Italia in 2002, received $8,000 for serving on an advisory board for Byk Gulden Italia in 2003, received $15,000 as a research grant from Byk Gulden Italia in 2003, and is going to receive $40,000 from Bayer P-Europe as a research grant for studies in ₁-antitrypsin deficiency.

REFERENCES

1. Mahadeva R, Shapiro SD. Chronic obstructive pulmonary disease. 3. Experimental animal models of pulmonary emphysema. Thorax 2002;57:908–914.[Abstract/Free Full Text]
2. Hogg JC, Senior RM. Chronic obstructive pulmonary disease. 2. Pathology and biochemistry of emphysema. Thorax 2002;57:830–834.[Abstract/Free Full Text]
3. Lee WJ, Downey GP. Leukocyte elastase. Am J Respir Crit Care Med 2001;164:896–904.[Free Full Text]
4. Snider GL. Experimental studies on emphysema and chronic bronchial injury. Eur J Respir Dis 1986;69:17–35.
5. Ying Q-L, Simon SR. DNA from bronchial secretions modulates elastase inhibition by ₁-proteinase inhibitor and oxidized secretory leukoprotease inhibitor. Am J Respir Cell Mol Biol 2000;23:506–513.[Abstract/Free Full Text]
6. Frommherz KJ, Faller B, Bieth JG. Heparin strongly decreases the rate of inhibition of neutrophil elastase by ₁-proteinase inhibitor. J Biol Chem 1991;266:15356–15362.[Abstract/Free Full Text]
7. Faller B, Cadène M, Bieth JG. Demonstration of a two step reaction mechanism for the inhibition of heparin-bound neutrophil elastase by ₁-proteinase inhibitor. Biochemistry 1993;32:9230–9235.[CrossRef][Medline]
8. Chan SCH, Shum DKY, Ip MSM. Sputum sol neutrophil elastase activity in bronchiectasis: differential modulation by syndecan-1. Am J Respir Crit Care Med 2003;168:192–198.[Abstract/Free Full Text]
9. Twumasi DY, Liener IE. Proteases from purulent sputum: purification and properties of the elastase and chymotrypsin-like enzymes. J Biol Chem 1977;252:1917–1926.[Abstract/Free Full Text]
10. Travis J, Salvesen GS. Human plasma proteinase inhibitors. Annu Rev Biochem 1983;52:655–709.[CrossRef][Medline]
11. Feller B, Mely Y, Gerard D, Bieth JG. Heparin-induced conformational change and activation of mucus proteinase inhibitor. Biochemistry 1992;31:8285–8290.[CrossRef][Medline]
12. Gottlieb DJ, Luisetti M, Stone PJ, Allegra L, Cantey-Kiser JM, Grassi C, Snider GL. Short-term supplementation therapy dose not affect elastin degradation in severe ₁-antitrypsin deficiency. Am J Respir Crit Care Med 2000;162:2069–2072.[Abstract/Free Full Text]
13. Stockley RA, Bayley DL, Unsal I, Dowson LJ. The effect of augmentation therapy on bronchial inflammation in ₁-antitrypsin deficiency. Am J Respir Crit Care Med 2002;165:1494–1498.[Abstract/Free Full Text]
14. Frevert CW, Kinsella MG, Vathanaprida C, Goodman RB, Baskin DG, Proudfoot A, Wells TNC, Wight TN, Martin TR. Binding of interleukin-8 to heparan sulfate and chondroitin sulfate in lung tissue. Am J Respir Cell Mol Biol 2003;28:464–472.[Abstract/Free Full Text]
15. Turino GM, Cantor JO. Hyaluronan in respiratory injury and repair. Am J Respir Crit Care Med 2003;167:1169–1175.[Free Full Text]

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