The role of secretory leukocyte proteinase inhibitor and elafin (elastase-specific inhibitor/skin-derived antileukoprotease) as alarm antiproteinases in inflammatory lung disease
© Current Science Ltd 2000
Received: 18 July 2000
Accepted: 7 August 2000
Published: 23 August 2000
Secretory leukocyte proteinase inhibitor and elafin are two low-molecular-mass elastase inhibitors that are mainly synthesized locally at mucosal sites. It is thought that their physicochemical properties allow them to efficiently inhibit target enzymes, such as neutrophil elastase, released into the interstitium. Historically, in the lung, these inhibitors were first purified from secretions of patients with chronic obstructive pulmonary disease and cystic fibrosis. This suggested that they might be important in controlling excessive neutrophil elastase release in these pathologies. They are upregulated by 'alarm signals' such as bacterial lipopolysaccharides, and cytokines such as interleukin-1 and tumor necrosis factor and have been shown to be active against Gram-positive and Gram-negative bacteria, so that they have joined the growing list of antimicrobial 'defensin-like' peptides produced by the lung. Their site of synthesis and presumed functions make them very attractive candidates as potential therapeutic agents under conditions in which the excessive release of elastase by neutrophils might be detrimental. Because of its natural tropism for the lung, the use of adenovirus-mediated gene transfer is extremely promising in such applications.
Cytokines form one of the major classes of chemical mediator responsible for initiating, regulating and terminating the inflammatory response. Their synthesis, switch-on and switch-off mechanisms and their mode of action are tightly regulated in what is now classically called a cytokine network. Indeed, early cytokines, such as interleukin-1 (IL-1) and tumor necrosis factor (TNF), are synthesized very quickly, within 1 h of the onset of inflammation, or in response to stimuli such as bacterial lipopolysaccharides (LPS).
These cytokines set in motion the migration of inflammatory cells such as neutrophils and monocytes, whose function is to eliminate the injurious agent and restore homeostasis. To migrate from the vascular space and gain access to the inflammatory site through the interstitium, it has been hypothesized that these inflammatory cells use a variety of proteases (such as neutrophil and monocyte/macrophage metalloproteases and elastases).
To contain the potential injurious effects of excess release of these proteases, the host secretes large amounts of antiproteinases, which also seem to have developed in a parallel network, consisting of `alarm' and `systemic' inhibitors, the latter being synthesized principally in the liver [such as α1-proteinase inhibitor (A1-Pi) and antichymotrypsin].
We are particularly interested in the former group, which include the two low-molecular-mass proteinase inhibitors of the ALP family, antileukoprotease (ALP), also known as secretory leukocyte proteinase inhibitor , mucus proteinase inhibitor or bronchial inhibitor (it will henceforth be referred to as SLPI), and elastase-specific inhibitor (ESI), also known as elafin or skin-derived antileukoprotease (SKALP) . They are synthesized and secreted locally at the site of injury and are produced in response to primary cytokines such as IL-1 and TNF; they might therefore be part of a first wave of local, inducible defense in the antiproteinases network . Although potentially secreted at many mucosal sites and sites of interface such as the skin , this review addresses mainly the role of SLPI and elafin in the lung.
Gene structure, protein and antiproteinase activity
SLPI is one of the two members of the ALP superfamily of proteinase inhibitors (the other being ESI/elafin; see below). The gene encoding SLPI [2.65 kilobases (kb) in length ] seems to be a relatively nonpolymorphic, stable gene that can be modulated at both the transcriptional and translational levels . Transfection studies with fusion elements composed of fragments of up to 1.2 kb of the 5' flanking region of the SLPI gene demonstrated a high promoter activity in a 131 base pair (bp) fragment (- 115 to + 16) relative to the transcription start site. Interestingly, Kikuchi et al  have recently delineated within this region a proximal 41-bp region that confers lung specificity for SLPI expression. It is a 11.7 kDa protein consisting of 107 amino acid residues  and comprising two domains. It contains 16 cysteine residues that form eight disulfide bridges . SLPI has been shown to inhibit human neutrophil elastase, cathepsin G, trypsin, chymotrypsin and chymase. Its major target is thought to be the human neutrophil elastase in view of its high affinity and kinetic constants (inhibition constant in the nanomolar range and k ass in the micromolar range) .
SLPI has been purified from different sources, including parotid, cervical, seminal and lung secretions .
Cell and tissue distribution
In lung, SLPI is produced in vitro by tracheal, bronchial, bronchiolar and type II alveolar cells, and by monocytes, alveolar macrophages and neutrophils [1,6]. It has also been shown to be produced in vivo by tracheal serous glands and bronchiolar Clara cells, and to be closely associated with elastin fibers in the alveolar interstitium . Its roles in inflammatory cells such as macrophages or neutrophils are uncertain but antibacterial or anti-inflammatory actions have been proposed (see below). Outside the lung, it is secreted in a variety of mucosal sites (leading to its alternative name, mucosal proteinase inhibitor ).
Gene structure, protein and antiproteinase activity
The neutrophil elastase inhibitor ESI/elafin (the other member of the ALP superfamily of proteinase inhibitors; reviewed in ) was first identified as a `non-SLPI' low-molecular-mass anti-elastase by Hochstrasser et al  and Kramps and Klasen , and further characterized by us in bronchial secretions  and by Wiedow et al and Molhuizen et al in the skin [10,11]. The sequence of the gene for ESI  showed that it is approx. 2.3 kb long, and is composed of three exons and two introns and containing typical 5' TATA and CAAT boxes, as well as 5' regulatory sequences such as activator protein-1 and nuclear factor-κB sites . Zhang et al demonstrated that a positive regulatory cis-element present in the region between -505 and -368 bp is responsible for the upregulation of the elafin gene in normal breast epithelial cells (reviewed in ). Further characterization will be needed to determine whether this region is tissue-specific or is also important for expression in lung cells. The molecule is composed of 117 amino acid residues, including a hydrophobic signal peptide of 22 residues. Elafin is part of a `four-disulfide' core protein family that has recently been termed the trappin family . Elafin can be divided into two domains, the carboxy-terminal domain containing the antiproteinase active site and the amino-terminal domain containing characteristic VKGQ sequences (in single-letter codes for amino acids). These sequences allow the elafin molecule to glue itself into polymers and bind other interstitial molecules through transglutamination . This feature could make elafin maximally effective as a tissue-bound inhibitor as opposed to A1-Pi, which is present in large amounts in the circulation. SLPI has also been suggested to have a locally protective role against neutrophilic damage, presumably because of its small size and negative charge. The elafin molecule shows a 40% homology with the SLPI molecule and the active sites of both inhibitors are very similar.
Like SLPI, elafin has a high content of cysteine residues, which are arranged in four disulfide bonds in the C-terminal proteinase-inhibiting region ; a partial crystal structure, not containing the VKGQ sequences (a major feature of the molecule) has recently been determined . Elafin has been shown to be more specific in its spectrum of inhibition than SLPI: it inhibits pig pancreatic elastase, human neutrophil elastase and proteinase-3 .
Cell and tissue distribution
As mentioned above, elafin was first demonstrated in the skin and in lung secretions but is also present at mucosal sites in many tissues. Its purification from sputum, its presence in tracheal biopsies and bronchoalveolar lavage from both normal subjects and patients , and its synthesis by Clara cells and type II cells indicate, as for SLPI, a tracheo-bronchioalveolar origin. It is present in highest concentrations in sputum (at approximately 10% of the concentration of SLPI). We have recently investigated its presence in the peripheral lung and have shown by immunohistochemistry that macrophages are a primary source (J-M Sallenave, unpublished data).
Regulation of SLPI and elafin
It has been shown that `alarm signals' such as bacterial LPS, IL-1, TNF, neutrophil elastase and defensins are able to switch on the production of these inhibitors [3,14,15]; conversely, anti-inflammatory and `remodelling' cytokines such as transforming growth factor-β can switch them off (this has been demonstrated at least for SLPI ). By comparison, `systemic inhibitors' such as A1-Pi and antichymotrypsin are upregulated mainly by a later wave of cytokines such as those of the IL-6 family (IL-6 and oncostatin M), suggesting that these different inhibitors might be physiologically involved at discrete and different time points as well as different locations. Interestingly, interferon-γ was shown to inhibit the LPS-induced SLPI upregulation in macrophages, suggesting a regulation of SLPI at the interface between innate and adaptive immunity .
Role of SLPI and elafin in innate immunity
Role of SLPI and ESI/elafin in lung pathophysiology
As mentioned above, SLPI and elafin were first isolated in the lung from sputum secretions from patients with chronic obstructive pulmonary disease (COPD) as the most abundant elastase inhibitors. It followed that one of their roles might be in controlling excess elastase release from the neutrophils present in these secretions.
COPD/emphysema and cystic fibrosis
Acute respiratory distress syndrome (ARDS) and pneumonia
In ARDS, both SLPI and elafin are markedly increased in bronchoalveolar lavage fluid  compared with control subjects. Interestingly, SLPI BAL levels were positively correlated with the multiple organ failure score (MOFS, see Fig. 2), suggesting that antiproteases such as SLPI might be released in this pathology as an aborted and unsuccessful attempt to control injury. Furthermore, in patients with pneumonia, SLPI levels were found to be increased in serum , which is consistent with the hypothesis that these inhibitors are `alarm inhibitors' whose role might be relevant at the onset of the inflammatory process.
Although still a contentious issue, emerging evidence suggests that serine proteases might be important in the pathophysiology of asthma. Mast cell and leukocyte serine protease levels are elevated in the airways of asthmatic patients and one study has shown that patients with A1-Pi deficiency develop asthma more readily . Although studies measuring SLPI and elafin in asthmatic patients are scarce, animal models of allergic diseases have shown SLPI to be of benefit in inhibiting both early and late phase events .
Therapeutic potential of SLPI and elafin
Use of recombinant proteins
A1-Pi protein replacement therapy is actively being pursued in patients with severe A1-Pi deficiency and COPD. Although definite results from these studies are eagerly awaited, low-molecular-mass protease inhibitors such as SLPI and elafin have the advantages of size, diffusibility, and possible anchorage to the interstitium (see above), which is where the action is. Although no studies in patients have been reported with the use of recombinant elafin, the use of SLPI in normal subjects and in cystic fibrosis patients has been investigated. Unfortunately, these studies have been hampered by a short half-life and poor accessibility of the recombinant product to diseased areas in cystic fibrosis patients (reviewed in ).
Although still in its infancy compared with pharmacological methods of drug delivery, gene therapy might be advantageous, owing to its less transient nature. Because of its natural tropism for the lung, adenovirus could be the vector of choice for gene therapy applications. Historically, most of these applications have been directed in the lung at chronic pathologies such as cystic fibrosis or cancer. This is by no means the best case, given the chronic nature of these pathologies in which a repeated administration of vectors would be necessary to achieve adequate levels of therapeutic transgenes. Unfortunately, the immune response directed against the currently available `first and second generation' adenovirus vectors currently precludes their use in such settings. However, the generation of `gutless' vectors (which express almost no viral genes) has shown to drastically improve the duration of transgene expression in rodent models . This type of vector is an important feature of adenovirus gene therapy techniques, but its generation remains technically challenging at present.
However, the constructs currently available are already useful for the study of experimental models in which immune responses are less of a concern and that mimic a clinical setting in which a therapeutic opportunity might exist between the presentation of patients and the development of irreversible acute inflammation. Such situations could include lung infections leading to pneumonia, ARDS or sepsis.
Indeed, success has been achieved in animal models of lung infections and endotoxemia in rodents, by using adenovirus vectors expressing molecules such as IL-12 and TNF (lung infections) and IL-10 (endotoxemia) [27–29]. An important and useful feature of adenovirus administration is the compartmentalization of transgenes administered in the lung, which thereby prevents unwanted systemic effects of potentially toxic molecules such as TNF. Because of the previously mentioned characteristics of SLPI and elafin as `alarm molecules' involved in the regulation of early events in the inflammatory process, the overexpression of these inhibitors would be of benefit in combating bacterial infections and their inflammatory sequelae. Our own studies have shown that the overexpression of elafin with an adenovirus vector (AdmCMV-elafin, in which CMV stands for cytomegalovirus)  significantly improved acute lung injury induced by Pseudomonas aeruginosa in C57/Bl6 mice (AJ Simpson and J-M Sallenave, unpublished data). The exact mechanism of the protection conferred by the elafin molecule is unclear at present and is still under investigation, but it might involve a dual mode of action, by killing the pathogens while dampening down the unwanted effects of neutrophil histotoxic molecules such as elastase.
SLPI and elafin characteristics
Four disulfide core
Four disulfide core/trappin
Molecular mass (kDa)
Spectrum of protease inhibition
HNE, trypsin, chymotrypsin, tryptase,
HNE, pig pancreatic elastase, proteinase-3
chymase, cathepsin G
Cell and tissue distribution in lung
Tracheal, bronchial, Clara, alveolar type II cells,
Tracheal, Clara, alveolar type II cells,
monocytes, alveolar macrophages, neutrophils
LPS, IL-1, TNF, HNE
IL-1, TNF, HNE
Antimicrobial, antiviral, anti-HNE, inhibition of
monocyte inflammatory potential
Gene therapy potential
Currently being explored
Adenovirus-elafin protects against lung injury
I acknowledge the support of the Salvesen Emphysema Research Trust, MRC-UK, the Wellcome Trust, the Scottish Hospital Endowment Research Trust, the British Lung Foundation, the Canadian Cystic Fibrosis Foundation and MRC-Canada. I thank the other members of my group, particularly Dr GA Cunningham, Dr PT Reid, Dr AJ Simpson, Mr ME Marsden and Professor C Haslett, for their involvement with the published work on SLPI and elafin, and also Professor J Gauldie, Dr SC Donnelly for his help in the study of acute lung injury, and Dr K Farmer for reviewing this manuscript.
- Stolk J, Hiemstra P: Recombinant SLPI: emphysema and asthma. In Emphysema and Infection. Edited by Stockley RA. Basel: Birkhauser; 1999,:55–67.Google Scholar
- Schalkwijk J, Wiedow O, Hirose S: The trappin gene family: proteins defined by an N-terminal transglutaminase substrate domain and a C-terminal four-disulphide core. Biochem J 1999, 340:569–577.View ArticlePubMedPubMed CentralGoogle Scholar
- Sallenave J-M, Schulmann J, Crosley J, Jordana M, Gauldie J: Regulation of secretory leukocyte proteinase inhibitor (SLPI) and elastase-specific inhibitor (ESI/elafin) in human airway epithelial cells by cytokines and neutrophilic enzymes. Am J Respir Cell Mol Biol 1994, 11:733–741.View ArticlePubMedGoogle Scholar
- Kikuchi T, Abe T, Satoh K, Narumi K, Sakai T, Abe S, Shindoh S, Matsushima K, Nukiwa T: cis-acting region associated with lung-cell specific expression of the secretory leukoprotease inhibitor gene. Am J Respir Cell Mol Biol 1997, 17:361–367.View ArticlePubMedGoogle Scholar
- Boudier C, Bieth JG: The proteinase-mucus proteinase inhibitor binding stoichiometry. J Biol Chem 1992, 267:4370–4375.PubMedGoogle Scholar
- Sallenave J-M, Si-Tahar M, Cox G, Chignard M, Gauldie J: Secretory leukocyte proteinase inhibitor is a major leukocyte elastase inhibitor in human neutrophils. J Leukoc Biol 1997, 61:695–702.PubMedGoogle Scholar
- Hochstrasser K, Albrecht GJ, Schonberger G, Rasche B, Lempart K: An elastase-specific inhibitor from human bronchial mucus. Isolation and characterization. Hoppe-Seyler's Z Physiol Chem 1981, 362:1369–1375.View ArticlePubMedGoogle Scholar
- Kramps JA, Klasen EC: Characterization of a low molecular weight anti-elastase isolated from human bronchial secretion. Exp Lung Res 1985, 9:151–165.View ArticlePubMedGoogle Scholar
- Sallenave JM, Ryle AP: Purification and characterization of elastase-specific inhibitor. Sequence homology with mucus proteinase inhibitor. Biol Chem Hoppe-Seyler 1991, 372:13–21.View ArticlePubMedGoogle Scholar
- Wiedow O, Schroder J, Gregory H, Young JA, Christophers E: Elafin: an elastase-specific inhibitor of human skin. Purification, characterization, and complete amino acid sequence. J Biol Chem 1990, 265:14791–14795.PubMedGoogle Scholar
- Molhuizen HOF, Alkemade HAC, Zeeuwen PLJM, de Johgh GJ, Wieringa B, Schalkwijk J: SKALP/elafin: an elastase inhibitor from cultured human keratinocytes. Purification, cDNA sequence, and evidence for transglutaminase cross-linking. J Biol Chem 1993, 268:12028–12032.PubMedGoogle Scholar
- Sallenave J-M, Silva A: Characterization and gene sequence of the precursor of elafin, an elastase-specific inhibitor in bronchial secretions. Am J Respir Cell Mol Biol 1993, 8:439–445.View ArticlePubMedGoogle Scholar
- Sallenave J-M, Donnelly SC, Grant IS, Robertson C, Gauldie J, Haslett C: Secretory leukocyte proteinase inhibitor is preferentially increased in patients with acute respiratory distress syndrome. Eur Respir J 1999, 13:1029–1036.View ArticlePubMedGoogle Scholar
- Van Wetering S, van der Linden AC, van Sterkenburg MA, de Boer WI, Kuijpers AL, Schalkwijk J, Hiemstra PS: Regulation of SLPI and elafin release from bronchial epithelial cells by neutrophil defensins. Am J Physiol Lung Cell Mol Physiol 2000, 278:L51-L58.PubMedGoogle Scholar
- Jin FY, Nathan CF, Radzioch D, Ding A: Lipopolysaccharide-related stimuli induce expression of the secretory leukocyte protease inhibitor, a macrophage-derived lipopolysaccharide inhibitor. Infect Immun 1998, 66:2447–2452.PubMedPubMed CentralGoogle Scholar
- Jaumann F, Elssner A, Mazur G, Dobmann S, Vogelmeier C: Transforming growth factor-beta1 is a potent inhibitor of secretory leukoprotease inhibitor expression in a bronchial epithelial cell line. Eur Respir J 2000, 15:1052–1057.View ArticlePubMedGoogle Scholar
- Jin FY, Nathan C, Radzioch D, Ding A: Secretory leukocyte protease inhibitor: a macrophage product induced by and antagonistic to bacterial lipopolysaccharide. Cell 1997, 88:417–426.View ArticlePubMedGoogle Scholar
- Simpson AJ, Maxwell AI, Govan JRW, Haslett C, Sallenave JM: Elafin (elastase-specific inhibitor) has anti-microbial activity against Gram-positive and Gram-negative respiratory pathogens. FEBS Lett 1999, 452:309–313.View ArticlePubMedGoogle Scholar
- Zhang Y, DeWitt DL, McNeely TB, Wahl SM, Wahl LM: Secretory leukocyte protease inhibitor suppresses the production of monocyte prostaglandin H synthase-2, prostaglandin E2, and matrix metalloproteinases. J Clin Invest 1997, 199:894–900.View ArticleGoogle Scholar
- Lentsch AB, Jordan AJ, Czermak BJ, Diehl KM, Younkin EM, Sarma V, Ward PA: Inhibition of NF-kappaB activation and augmentation of Ikappabeta by secretory leukocyte protease inhibitor during lung inflammation. Am J Pathol 1999, 154:239–247.View ArticlePubMedPubMed CentralGoogle Scholar
- Morrison HM, Kramps JA, Burnett D, Stockley RA: Lung lavage fluid from patients with alpha-1 proteinase inhibitor deficiency or chronic obstructive bronchitis: anti-elastase function and cell profile. Clin Sci 1987, 72:373–381.View ArticlePubMedGoogle Scholar
- Stockley RA, Morrison HM, Smith S, Tetley T: Low molecular mass bronchial proteinase inhibitor and alpha-1 proteinase inhibitor in sputum and bronchoalveolar lavage. Hoppe-Seyler's Z. Physiol. Chem 1984, 365:587–595.View ArticlePubMedGoogle Scholar
- Asano S, Kida K, Koyama T, Wada H, Izawa Y, Hosoda K, Masuda K, Suzuki Y: A morphological study of lung secretory leukoprotease inhibitor in pneumonia. Am J Respir Crit Care Med 1995, 151:1576–1581.View ArticlePubMedGoogle Scholar
- Eden E, Mitchell D, Mehlman B, Khouli H, Nejat M, Grieco MH, Turino GM: Atopy, asthma, and emphysema in patients with severe alpha-1 antitrypsin deficiency. Am J Respir Crit Care Med 1997, 156:68–74.View ArticlePubMedGoogle Scholar
- Wright CD, Havill AM, Middleton SC, Kashem MA, Lee PA, Dripps DJ, O'Riordan TG, Bevilacqua MP, Abraham WM: Secretory leukocyte protease inhibitor prevents allergen-induced pulmonary responses in animal models of asthma. J Pharmacol Exp Ther 1999, 289:1007–1014.PubMedGoogle Scholar
- Morral N, O'Neal W, Rice K, Leland M, Kaplan J, Piedra PA, Zhou H, Parks RJ, Velji R, Aguilar-Cordova E, Wadsworth S, Graham FL, Kochanek S, Carey KD, Beaudet AL: Administration of helper-dependent adenoviral vectors and sequential delivery of different vector serotype for long-term liver-directed gene transfer in baboons. Proc Natl Acad Sci USA 1999, 96:12816–12821.View ArticlePubMedPubMed CentralGoogle Scholar
- Greenberger MJ, Kunkel SL, Strieter RM, Lukacs NW, Bramson J, Gauldie J, Graham FL, Hitt M, Danforth JM, Standiford TJ: IL-12 gene therapy protects mice in lethal Klebsiella pneumonia. J Immunol 1996, 157:3006–3012.PubMedGoogle Scholar
- Kolls J, Lei D, Nelson S, Summer WR, Greenberg S, Beutler B: Adenovirus-mediated blockade of tumor necrosis factor in mice protects against endotoxic shock yet impairs pulmonary host defense. J Infect Dis 1995, 171:570–575.View ArticlePubMedGoogle Scholar
- Xing Z, Ohkawara Y, Jordana M, Graham FL, Gauldie J: Adenoviral vector-mediated interleukin-10 expression in vivo: intramuscular gene transfer inhibits cytokine responses in endotoxemia. Gene Ther 1997, 4:140–149.View ArticlePubMedGoogle Scholar
- Sallenave J-M, Xing Z, Simpson AJ, Graham FL, Gauldie J: Adenovirus-mediated expression of an elastase-specific inhibitor (elafin): a comparison of different promoters. Gene Therapy 1998, 5:352–360.View ArticlePubMedGoogle Scholar