Protease inhibitors derived from elafin and SLPI and engineered to have enhanced specificity towards neutrophil serine proteases

The secretory leukocyte protease inhibitor (SLPI), elafin, and its biologically active precursor trappin‐2 are endogeneous low‐molecular weight inhibitors of the chelonianin family that control the enzymatic activity of neutrophil serine proteases (NSPs) like elastase, proteinase 3, and cathepsin G. These inhibitors may be of therapeutic value, since unregulated NSP activities are linked to inflammatory lung diseases. However SLPI inhibits elastase and cathepsin G but not proteinase 3, while elafin targets elastase and proteinase 3 but not cathepsin G. We have used two strategies to design polyvalent inhibitors of NSPs that target all three NSPs and may be used in the aerosol‐based treatment of inflammatory lung diseases. First, we fused the elafin domain with the second inhibitory domain of SLPI to produce recombinant chimeras that had the inhibitory properties of both parent molecules. Second, we generated the trappin‐2 variant, trappin‐2 A62L, in which the P1 residue Ala is replaced by Leu, as in the corresponding position in SLPI domain 2. The chimera inhibitors and trappin‐2 A62L are tight‐binding inhibitors of all three NSPs with subnanomolar K_is, similar to those of the parent molecules for their respective target proteases. We have also shown that these molecules inhibit the neutrophil membrane‐bound forms of all three NSPs. The trappin‐2 A62L and elafin‐SLPI chimeras, like wild‐type elafin and trappin‐2, can be covalently cross‐linked to fibronectin or elastin by a tissue transglutaminase, while retaining their polypotent inhibition of NSPs. Therefore, the inhibitors described herein have the appropriate properties to be further evaluated as therapeutic anti‐inflammatory agents.     

Secretory leukocyte protease inhibitor (SLPI) is, like its homologue trappin-2 (pre-elafin), a transglutaminase substrate

Human lungs contain secretory leukocyte protease inhibitor (SLPI), elafin and its biologically active precursor trappin-2 (pre-elafin). These important low-molecular weight inhibitors are involved in controlling the potentially deleterious proteolytic activities of neutrophil serine proteases including elastase, proteinase 3 and cathepsin G. We have shown previously that trappin-2, and to a lesser extent, elafin can be linked covalently to various extracellular matrix proteins by tissue transglutaminases and remain potent protease inhibitors. SLPI is composed of two distinct domains, each of which is about 40% identical to elafin, but it lacks consensus transglutaminase sequence(s), unlike trappin-2 and elafin. We investigated the actions of type 2 tissue transglutaminase and plasma transglutaminase activated factor XIII on SLPI. It was readily covalently bound to fibronectin or elastin by both transglutaminases but did not compete with trappin-2 cross-linking. Cross-linked SLPI still inhibited its target proteases, elastase and cathepsin G. We have also identified the transglutamination sites within SLPI, elafin and trappin-2 by mass spectrometry analysis of tryptic digests of inhibitors cross-linked to mono-dansyl cadaverin or to a fibronectin-derived glutamine-rich peptide. Most of the reactive lysine and glutamine residues in SLPI are located in its first N-terminal elafin-like domain, while in trappin-2, they are located in both the N-terminal cementoin domain and the elafin moiety. We have also demonstrated that the transglutamination substrate status of the cementoin domain of trappin-2 can be transferred from one protein to another, suggesting that it may provide transglutaminase-dependent attachment properties for engineered proteins. We have thus added to the corpus of knowledge on the biology of these potential therapeutic inhibitors of airway proteases.     

Multifaceted roles of human elafin and secretory leukocyte proteinase inhibitor (SLPI), two serine protease inhibitors of the chelonianin family

Elafin and SLPI are low-molecular weight proteins that were first identified as protease inhibitors in mucous fluids including lung secretions, where they help control excessive proteolysis due to neutrophil serine proteases (elastase, proteinase 3 and cathepsin G). Elafin and SLPI are structurally related in that both have a fold with a four-disulfide core or whey acidic protein (WAP) domain responsible for inhibiting proteases. Elafin is derived from a precursor, trappin-2 or pre-elafin, by proteolysis. Trappin-2, which is itself a protease inhibitor, has a unique N-terminal domain that enables it to become cross-linked to extracellular matrix proteins by transglutaminase(s). SLPI and elafin/trappin-2 are attractive candidates as therapeutic molecules for inhibiting neutrophil serine proteases in inflammatory lung diseases. Hence, they have become the WAP proteins most studied over the last decade. This review focuses on recent findings revealing that SLPI and elafin/trappin-2 have many biological functions as diverse as anti-bacterial, anti-fungal, anti-viral, anti-inflammatory and immuno-modulatory functions, in addition to their well-recognized role as protease inhibitors.     

Novel roles of protease inhibitors in infection and inflammation

The local balance between proteinase inhibitors and proteinases determines local proteolytic activity. Various studies have demonstrated the importance of serine proteinase inhibitors in regulating the activity of serine proteinases that are released by leucocytes during inflammation. Recently it has been shown that these inhibitors may also display functions that are distinct from those associated with the inhibition of leucocyte-derived proteinases. In this review the results of selected studies focusing on three inhibitors of neutrophil elastase, i.e. alpha(1)-proteinase inhibitor, secretory leucocyte proteinase inhibitor and elafin, are presented, with the aim of illustrating their possible involvement in the regulation of inflammation, host defence against infection, tissue repair and extracellular matrix synthesis.     

Regulation of SLPI and elafin release from bronchial epithelial cells by neutrophil defensins

Secretory leukocyte proteinase inhibitor (SLPI) is a serine proteinase inhibitor that is produced locally in the lung by cells of the submucosal bronchial glands and by nonciliated epithelial cells. Its main function appears to be the inhibition of neutrophil elastase (NE). Recently, NE was found to enhance SLPI mRNA levels while decreasing SLPI protein release in airway epithelial cells. Furthermore, glucocorticoids were shown to increase both constitutive and NE-induced SLPI mRNA levels. In addition to NE, stimulated neutrophils also release alpha-defensins. Defensins are small, antimicrobial polypeptides that are found in high concentrations in purulent secretions of patients with chronic airway inflammation. Like NE, defensins induce interleukin-8 production in airway epithelial cells. This induction is sensitive to inhibition by the glucocorticoid dexamethasone and is prevented in the presence of alpha(1)-proteinase inhibitor. The aim of the present study was to investigate the effect of defensins on the production of SLPI and the related NE inhibitor elafin/SKALP in primary bronchial epithelial cells (PBECs). Defensins significantly increase SLPI protein release by PBECs in a time- and dose-dependent fashion without affecting SLPI mRNA synthesis. In the presence of alpha(1)-proteinase inhibitor, the defensin-induced SLPI protein release is further enhanced, but no effect was observed on SLPI mRNA levels. Dexamethasone did not affect SLPI protein release from control or defensin-treated PBECs. In addition, we observed a constitutive release of elafin/SKALP by PBECs, but this was not affected by defensins. The present results suggest a role for defensins in the dynamic regulation of the antiproteinase screen in the lung at sites of inflammation.     

SLPI and trappin-2 as therapeutic agents to target airway serine proteases in inflammatory lung diseases: current and future directions

It is now clear that NSPs (neutrophil serine proteases), including elastase, Pr3 (proteinase 3) and CatG (cathepsin G) are major pathogenic determinants in chronic inflammatory disorders of the lungs. Two unglycosylated natural protease inhibitors, SLPI (secretory leucocyte protease inhibitor) and elafin, and its precursor trappin-2 that are found in the lungs, have therapeutic potential for reducing the protease-induced inflammatory response. This review examines the multifaceted roles of SLPI and elafin/trappin-2 in the context of their possible use as inhaled drugs for treating chronic lung diseases such as CF (cystic fibrosis) and COPD (chronic obstructive pulmonary disease).     

The role of secretory leukocyte proteinase inhibitor and elafin (elastase-specific inhibitor/skin-derived antileukoprotease) as alarm antiproteinases in inflammatory lung disease

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.     

WAP domain proteins as modulators of mucosal immunity

WAP (whey acidic protein) is an important whey protein present in milk of mammals. This protein has characteristic domains, rich in cysteine residues, called 4-DSC (four-disulfide core domain). Other proteins, mainly present at mucosal surfaces, have been shown to also possess these characteristic WAP-4-DSC domains. The present review will focus on two WAP-4-DSC containing proteins, namely SLPI (secretory leucocyte protease inhibitor) and trappin-2/elafin. Although first described as antiproteases able to inhibit in particular host neutrophil proteases [NE (neutrophil elastase), cathepsin-G and proteinase-3] and as such, able to limit maladaptive tissue damage during inflammation, it has become apparent that these molecules have a variety of other functions (direct antimicrobial activity, bacterial opsonization, induction of adaptive immune responses, promotion of tissue repair, etc.). After providing information about the 'classical' antiproteasic role of these molecules, we will discuss the evidence pertaining to their pleiotropic functions in inflammation and immunity.     

Elafin, an elastase-specific inhibitor, is cleaved by its cognate enzyme neutrophil elastase in sputum from individuals with cystic fibrosis

Elafin is a neutrophil serine protease inhibitor expressed in lung and displaying anti-inflammatory and anti-bacterial properties. Previous studies demonstrated that some innate host defense molecules of the cystic fibrosis (CF) and chronic obstructive pulmonary disease airways are impaired due to increased proteolytic degradation observed during lung inflammation. In light of these findings, we thus focused on the status of elafin in CF lung. We showed in the present study that elafin is cleaved in sputum from individuals with CF. Pseudomonas aeruginosa-positive CF sputum, which was found to contain lower elafin levels and higher neutrophil elastase (NE) activity compared with P. aeruginosa-negative samples, was particularly effective in cleaving recombinant elafin. NE plays a pivotal role in the process as only NE inhibitors are able to inhibit elafin degradation. Further in vitro studies demonstrated that incubation of recombinant elafin with excess of NE leads to the rapid cleavage of the inhibitor. Two cleavage sites were identified at the N-terminal extremity of elafin (Val-5-Lys-6 and Val-9-Ser-10). Interestingly, purified fragments of the inhibitor (Lys-6-Gln-57 and Ser-10-Gln-57) were shown to still be active for inhibiting NE. However, NE in excess was shown to strongly diminish the ability of elafin to bind lipopolysaccharide (LPS) and its capacity to be immobilized by transglutamination. In conclusion, this study provides evidence that elafin is cleaved by its cognate enzyme NE present at excessive concentration in CF sputum and that P. aeruginosa infection promotes this effect. Such cleavage may have repercussions on the innate immune function of elafin.     

Susceptibility of lung epithelium to neutrophil elastase: protection by native inhibitors

The development of emphysema is thought to be due to an imbalance of proteases (especially neutrophil elastase [NE]) and antiproteases with loosening of the respiratory epithelium as an early event. We investigated the effect of NE on respiratory epithelial cell adherence in vitro , in the presence of varying concentrations and combinations of native inhibitors, alpha-1-proteinase inhibitor (PI) and secretory leukoprotease inhibitor (SLPI). SLPI was two to 12 times more effective than PI at preventing the effects of NE, especially when enzyme:inhibitor ratios were almost equivalent. Even when the concentration of SLPI was only 10% of the total (as in normal peripheral lung secretions), it gave greater protection than PI alone. This suggests that SLPI plays an important role in controlling neutrophil elastaseinduced inflammation and tissue damage.     

The presence of elafin, SLPI, IL1-RA and STNFalpha RI in head and neck squamous cell carcinomas and their relation to the degree of tumour differentiation

Biopsy samples of head and neck carcinomas were investigated with regard to elafin, secretory leukocyte protease inhibitor (SLPI), interleukin 1-receptor antagonist [(IL)1-RA] and soluble tumour necrosis factor alpha receptor antagonist (STNFalpha RI). SLPI and elafin are serine protease inhibitors produced in the serous cells of the upper respiratory airways and in the keratinocytes, respectively. We have now found the presence of elafin and SLPI in squamous cell carcinomas of the upper respiratory tract (tonsillar, hypopharyngeal, tongue, mouth floor, gingival and laryngeal cancer). Significantly higher amounts of SLPI and elafin are present in well-differentiated and moderately differentiated tumours than in poorly differentiated tumours (p < 0.0001 and p < 0.0015). Tumour necrosis factor-alpha and IL-1beta have been shown to stimulate the production of SLPI and elafin. Since these cytokines can both be difficult to detect, we chose to study their inhibitors, STNFalpha RI and IL1-RA, instead. IL1-RA was expressed in highly differentiated tumours as well as in poorly differentiated ones. No significant difference was seen between the groups. STNFalpha RI was only found in very small amounts, sparsely distributed in the tumours, and was not related to the degree of differentiation.     

Secretory leucoprotease inhibitor prevents lipopolysaccharide-induced IkappaBalpha degradation without affecting phosphorylation or ubiquitination

Secretory leucoprotease inhibitor (SLPI) is a non-glycosylated protein produced by epithelial cells, macrophages, and neutrophils and was initially identified as a serine protease inhibitor of the neutrophil proteases elastase and cathepsin G. In addition to its antiprotease activity, SLPI has been shown to exhibit anti-inflammatory properties including down-regulation of tumor necrosis factor-alpha expression by lipopolysaccharide (LPS) in monocytes, inhibition of NF-kappaB activation by IgG immune complexes in a rat model of acute lung injury, and prevention of human immunodeficiency virus infectivity in monocytic cells via as yet unidentified mechanisms. In this report we have shown that SLPI prevents LPS-induced NF-kappaB activation by inhibiting degradation of IkappaBalpha without affecting the LPS-induced phosphorylation and ubiquitination of IkappaBalpha. We have also demonstrated that SLPI prevents LPS-induced interleukin-1 receptor-associated kinase and IkappaBbeta degradation. In addition, we have demonstrated that oxidized SLPI, a variant of SLPI that has diminished antiprotease activity, cannot prevent LPS-induced NF-kappaB activation or Inhibitor kappaB alpha/beta degradation indicating that the anti-inflammatory effect of SLPI on the LPS-signaling pathway is dependent on its antiprotease activity. These results suggest that SLPI may be inhibiting proteasomal degradation of NF-kappaB regulatory proteins, an effect that is dependent on the antiprotease activity of SLPI.     

Host defense effector molecules in mucosal secretions

Mucosal secretions contain a range of defense effector molecules including antimicrobial peptides and proteinase inhibitors. These molecules play a central role in host defense against infection, and in a variety of immune and inflammatory reactions. The aim of this study was to analyze the levels of neutrophil defensins, the cathelicidin hCAP-18/LL-37, and the proteinase inhibitors secretory leukocyte proteinase inhibitor, SKALP/elafin and cystatin M/E in various mucosal secretions and urine. We show here that especially seminal plasma is characterized by high concentrations of hCAP-18/LL-37, SLPI, SKALP/elafin and cystatin M/E. The results of this study demonstrate that each mucosal secretion is characterized by a unique profile of effector molecules, which may supply individual mucosal secretions with specific properties related to the control of local infection and inflammation.     

Mutants of plasminogen activator inhibitor-1 designed to inhibit neutrophil elastase and cathepsin G are more effective in vivo than their endogenous inhibitors

Neutrophil elastase and cathepsin G are abundant intracellular neutrophil proteinases that have an important role in destroying ingested particles. However, when neutrophils degranulate, these proteinases are released and can cause irreparable damage by degrading host connective tissue proteins. Despite abundant endogenous inhibitors, these proteinases are protected from inhibition because of their ability to bind to anionic surfaces. Plasminogen activator inhibitor type-1 (PAI-1), which is not an inhibitor of these proteinases, possesses properties that could make it an effective inhibitor of neutrophil proteinases if its specificity could be redirected. PAI-1 efficiently inhibits surface-sequestered proteinases, and it efficiently mediates rapid cellular clearance of PAI-1-proteinase complexes. Therefore, we examined whether PAI-1 could be engineered to inhibit and clear neutrophil elastase and cathepsin G. By introducing specific mutations in the reactive center loop of wild-type PAI-1, we generated PAI-1 mutants that are effective inhibitors of both proteinases. Kinetic analysis shows that the inhibition of neutrophil proteinases by these PAI-1 mutants is not affected by the sequestration of neutrophil elastase and cathepsin G onto surfaces. In addition, complexes of these proteinases and PAI-1 mutants are endocytosed and degraded by lung epithelial cells more efficiently than either the neutrophil proteinases alone or in complex with their physiological inhibitors, alpha1-proteinase inhibitor and alpha1-antichymotrypsin. Finally, the PAI-1 mutants were more effective in reducing the neutrophil elastase and cathepsin G activities in an in vivo model of lung inflammation than were their physiological inhibitors.     

Conversion of proepithelin to epithelins: roles of SLPI and elastase in host defense and wound repair

Increased leukocyte elastase activity in mice lacking secretory leukocyte protease inhibitor (SLPI) leads to impaired wound healing due to enhanced activity of TGFbeta and perhaps additional mechanisms. Proepithelin (PEPI), an epithelial growth factor, can be converted to epithelins (EPIs) in vivo by unknown mechanisms with unknown consequences. We found that PEPI and EPIs exert opposing activities. EPIs inhibit the growth of epithelial cells but induce them to secrete the neutrophil attractant IL-8, while PEPI blocks neutrophil activation by tumor necrosis factor, preventing release of oxidants and proteases. SLPI and PEPI form complexes, preventing elastase from converting PEPI to EPIs. Supplying PEPI corrects the wound-healing defect in SLPI null mice. Thus, SLPI/elastase act via PEPI/EPIs to operate a switch at the interface between innate immunity and wound healing.     

Adenoviral augmentation of elafin protects the lung against acute injury mediated by activated neutrophils and bacterial infection

During acute pulmonary infection, tissue injury may be secondary to the effects of bacterial products or to the effects of the host inflammatory response. An attractive strategy for tissue protection in this setting would combine antimicrobial activity with inhibition of human neutrophil elastase (HNE), a key effector of neutrophil-mediated tissue injury. We postulated that genetic augmentation of elafin (an endogenous inhibitor of HNE with intrinsic antimicrobial activity) could protect the lung against acute inflammatory injury without detriment to host defense. A replication-deficient adenovirus encoding elafin cDNA significantly protected A549 cells against the injurious effects of both HNE and whole activated human neutrophils in vitro. Intratracheal replication-deficient adenovirus encoding elafin cDNA significantly protected murine lungs against injury mediated by Pseudomonas aeruginosa in vivo. Genetic augmentation of elafin therefore has the capacity to protect the lung against the injurious effects of both bacterial pathogens resistant to conventional antibiotics and activated neutrophils.     

Selective role of PI3K delta in neutrophil inflammatory responses

Although members of the class I phosphoinositide 3-kinases (PI3Ks) have been implicated in neutrophil inflammatory responses, the contribution of the individual PI3K isoforms in neutrophil activation has not been tractable with the non-selective inhibitors, LY294002 and wortmannin. We have developed a novel series of PI3K inhibitors that is selective for PI3K delta, an isoform expressed predominantly in hematopoietic cells. In addition to being selective between members of class I PI3Ks, representatives of these inhibitors such as IC980033 and IC87114 did not inhibit any protein kinases tested. Utilizing these inhibitors we report here a novel role for PI3K delta in neutrophil activation. Inhibition of PI3K delta with IC980033 and IC87114 blocked both fMLP- and TNF1 alpha-induced neutrophil superoxide generation and elastase exocytosis. The PI3K delta inhibitor IC87114 also blocked TNF1 alpha-stimulated elastase exocytosis from neutrophils in a mouse model of inflammation. To our knowledge, this is the first in vivo efficacy demonstration of a PI3K delta inhibitor in an animal model. Inhibition of PI3K delta, however, had no effect on in vitro neutrophil bactericidal activity and Fc gamma R-stimulated superoxide generation. Thus, PI3K delta plays an essential role in certain signaling pathways of neutrophil activation and appears to be an attractive target for the development of an anti-inflammatory therapeutic.