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Keywords:

  • cathepsin G;
  • cytokines;
  • elastase;
  • proteinase 3;
  • receptor;
  • TNF-α

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Conversion of cytokines and growth factors
  5. Proteinase 3 converts TNF-α
  6. Proteinase 3 activates IL-1β
  7. Proteinase 3 activates IL-18
  8. HLE activates EGFR
  9. HLE may activate toll-like receptor-4
  10. Cathepsin G activates protease-activated receptor-4
  11. Caspase-like activity
  12. Shedding of receptors and cytokines
  13. Therapeutic approaches
  14. Conclusion
  15. Conflict of interest statement
  16. Acknowledgements
  17. References

Abstract.  Wiedow O, Meyer-Hoffert U (University Kiel, Kiel, Germany; and Karolinska Institute, Stockholm, Sweden). Neutrophil serine proteases: potential key regulators of cell signalling during inflammation (Review). J Intern Med 2005; 257: 319–328.

The serine proteases cathepsin G, human leucocyte elastase and proteinase 3 are major contents of neutrophils and are released at sites of inflammation. The common picture of their function was that they do not degrade extracellular proteins specifically. Recent studies provided evidence that these proteases are able to activate specifically pro-inflammatory cytokines and lead to the activation of different receptors. Neutrophil serine proteases might therefore be important regulators of inflammatory processes and are interesting targets for new therapeutic approaches against inflammatory disorders. This review summarizes the current knowledge on the regulation of cell signalling by neutrophil serine proteases.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Conversion of cytokines and growth factors
  5. Proteinase 3 converts TNF-α
  6. Proteinase 3 activates IL-1β
  7. Proteinase 3 activates IL-18
  8. HLE activates EGFR
  9. HLE may activate toll-like receptor-4
  10. Cathepsin G activates protease-activated receptor-4
  11. Caspase-like activity
  12. Shedding of receptors and cytokines
  13. Therapeutic approaches
  14. Conclusion
  15. Conflict of interest statement
  16. Acknowledgements
  17. References

Neutrophil infiltration is a common pathological feature in acute inflammatory disorders. The primary function of neutrophils is the phagocytosis and eradication of microorganisms. Every doctor is aware of their crucial importance to protect the host from bacterial infections whilst looking at the blood count of a neutropenic patient. After being generated in the bone marrow the neutrophils circulate in the bloodstream. They adhere to the endothelium and migrate to the infected site where they release oxygen radicals and their internal constituents, which are stored in different granules [1]. The azurophil granules, also called primary granules, contain the serine proteases human leucocyte elastase (HLE), cathepsin G (CG) and proteinase 3 (PR3) in high concentrations around 1 pg enzyme per cell [2]. As azurophil granules undergo limited exocytosis in response to stimulation compared with the other neutrophil granules, it is believed that their primary function is to kill and degrade the engulfed microorganisms in the phagolysosome. Considering the acidic milieu within the phagolysosome it is questionable whether intralysosomal proteolysis is the only function of these enzymes, which have an activity optimum around neutral pH values (Table 1). Recently a novel mechanism has described how neutrophils release granule proteins and chromatin that together form extracellular fibres to bind and kill bacteria [3]. HLE is present in these fibres and plays a crucial role in targeting and cleaving bacterial virulence factors [4].

Table 1.  Overview of neutrophil serine proteases
 Human leucocyte elastaseCathepsin GProteinase 3
  1. SLPI, secretory leucocyte protease inhibitor; MNEI, monocyte neutrophil elastase inhibitor.

EC numberEC 3.4.21.37EC 3.4.21.20EC 3.4.21.76
Size218 amino acids235 amino acids222 amino acids
Mass (kDa)3028.529
pIpI > 9pI ∼ 12pI > 9
pH optimum8.0–8.5∼8.0∼7.5
Substrate specificityVal-Xaa > Ala-XaaAromatic acids in P1-positionSimilar to HLE, accepts basic amino acids in P1-position
Endogenous inhibitorsα1-proteinase inhibitorα1-proteinase inhibitorα1-proteinase inhibitor
α2-macroglobulinα1-antichymotrypsinα2-macroglobulin
SLPISLPI 
Elafin Elafin
MNEIMNEIMNEI

However, neutrophils are also present in inflammatory disorders without bacterial involvement. Their active enzymes are detectable at sites of inflammation even though protease inhibitors exist in plasma and in the tissue. For example, active HLE is detectable and can be quantified on the surface of psoriatic plaques in psoriasis, an inflammatory skin disorder with a typical neutrophil infiltrate into the epidermis. HLE activity corresponded to the level of inflammation and disappeared after successful treatment [5].

In recent years more and more attention has focused on the extracellular effects of neutrophil serine proteases. Whilst the interest aimed in the beginning more at the deleterious potential like their matrix-degrading activity, various studies have shown evidence that neutrophil serine proteases aim specifically at a variety of regulatory functions in local inflammatory processes. Like the manifold diseases where neutrophils contribute to the pathological picture, these studies focused on different organs, cells and molecules. The purpose of this review is to present the latest and most important work on neutrophil serine protease participation on cell signalling. The reader not familiar with this subject shall get an idea that these proteases are more an important regulatory tool in inflammatory processes rather than a nonspecific degradation machinery.

This review summarizes the knowledge that neutrophil serine proteases process cytokines and growth factors and stimulate various cellular receptors. Furthermore, it will discuss that neutrophil serine proteases can act as exogenous caspase-like enzymes and inactivate signalling receptors and cytokines selectively. Knowing the molecular function of neutrophil serine proteases is necessary to understand the pathology of inflammatory disorders and may help to find new therapeutic approaches in future.

Proteinase 3 converts TNF-α

  1. Top of page
  2. Abstract
  3. Introduction
  4. Conversion of cytokines and growth factors
  5. Proteinase 3 converts TNF-α
  6. Proteinase 3 activates IL-1β
  7. Proteinase 3 activates IL-18
  8. HLE activates EGFR
  9. HLE may activate toll-like receptor-4
  10. Cathepsin G activates protease-activated receptor-4
  11. Caspase-like activity
  12. Shedding of receptors and cytokines
  13. Therapeutic approaches
  14. Conclusion
  15. Conflict of interest statement
  16. Acknowledgements
  17. References

Enzymatic processing of cytokine and growth factor precursors after translation is a common principle of their activation and allows a fast response to various stimuli.

One of the most important cytokines during inflammation is TNF-α, which was demonstrated impressively by the therapeutic success of anti-TNF-α treatment in Crohn's disease, rheumatoid arthritis and psoriasis. TNF-α is produced as a membrane-bound pro-form, which needs to be cleaved proteolytically to be released in its major biological active form. The membrane-bound metalloproteinase TNF-α converting enzyme (TACE) is responsible for TNF-α processing. However, serine protease inhibitors suppressed the secretion of TNF-α from activated macrophages [6, 7]. Moreover, mice pretreated with the serine protease inhibitor a1-antitrypsin were not able to secrete TNF-α in response to d-galactosamine together with lipopolysaccharide (LPS) thus becoming fully protected against d-galactosamine/LPS-induced hepatitis [8]. PR3, but not HLE, is able to process TNF-αin vitro into its active form [9] (Fig. 1). Cleavage occurs between Val77 and Arg78 and differs from the TACE cleavage sites Val79 and Asp80 without affecting the biological activity significantly.

image

Figure 1. Cleavage of membrane bound pro-TNF-α either by the membrane-bound matrix metalloproteinase TNF-α converting enzyme (TACE) or extracellular serine-protease proteinase 3 (PR3) to its active soluble form.

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Although anti-TNF-α treatment is very successful in certain diseases like Crohn's disease and rheumatoid arthritis, it runs a certain risk of promoting infectious diseases like tuberculosis. It is tempting to speculate that a more selective inhibition of TNF-α by PR3 inhibitors could have a benefit in theses cases as the endogenous cleavage pathway by the cell-bound TACE is not affected.

Proteinase 3 activates IL-1β

  1. Top of page
  2. Abstract
  3. Introduction
  4. Conversion of cytokines and growth factors
  5. Proteinase 3 converts TNF-α
  6. Proteinase 3 activates IL-1β
  7. Proteinase 3 activates IL-18
  8. HLE activates EGFR
  9. HLE may activate toll-like receptor-4
  10. Cathepsin G activates protease-activated receptor-4
  11. Caspase-like activity
  12. Shedding of receptors and cytokines
  13. Therapeutic approaches
  14. Conclusion
  15. Conflict of interest statement
  16. Acknowledgements
  17. References

Another important pro-inflammatory cytokine is IL-1β, which participates in inflammatory diseases like rheumatoid arthritis [10], neuroinflammation [11] and many others. IL-1β, like TNF-α, is synthesized as an immature and inactive precursor [12, 13]. Conversion to the active form depends on cleavage by a cysteine proteinase, named caspase-1 or IL-1β-converting enzyme, which also may be important for transport of the mature protein across the cytoplasmic membrane [14–16]. Furthermore, the release of pro-IL-1β from activated monocytes has also been described [17, 18]. Caspase-1-deficient mice can still generate mature IL-1β in response to a local inflammatory stimulus [19] indicating that alternative processing of this cytokine in vivo. Activated neutrophils or purified PR3 released TNF-α and IL-1β from a stimulated human monocyte cell line independent of converting enzyme release (Fig. 2). In consideration that keratinocytes produce but are not able to process pro-IL-1β, one may suggest a requirement for extracellular processing particularly in the context of localized inflammatory processes.

image

Figure 2. Conversion of IL-1β and IL-18 either by IL-1 converting enzyme (ICE) or extracellular proteinase3 (PR3) to its active soluble form.

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Proteinase 3 activates IL-18

  1. Top of page
  2. Abstract
  3. Introduction
  4. Conversion of cytokines and growth factors
  5. Proteinase 3 converts TNF-α
  6. Proteinase 3 activates IL-1β
  7. Proteinase 3 activates IL-18
  8. HLE activates EGFR
  9. HLE may activate toll-like receptor-4
  10. Cathepsin G activates protease-activated receptor-4
  11. Caspase-like activity
  12. Shedding of receptors and cytokines
  13. Therapeutic approaches
  14. Conclusion
  15. Conflict of interest statement
  16. Acknowledgements
  17. References

Interleukin-18 is a member of the IL-1 cytokine family, which is known to be cleaved by caspase-1 to its active form. The inactive precursor of IL-18 was also identified as a substrate of PR3. The IL-18 precursor is constitutively expressed in primary human oral epithelial cells and several epithelial cell lines. When primed by IFN-γ and subsequently stimulated with PR3 in the presence of LPS, these cells release active IL-18 into the supernatant [20]. The appearance of active IL-18 was not due to cell leakage or death. The release of active IL-18 was independent of caspase-1 activity. Injection of mice with recombinant Fas ligand resulted in hepatic damage, which was IL-18-dependent. However, the same results were obtained in mice deficient in caspase-1 [21] implicating an alternative activation mode for IL-18 in vivo. It is quite likely that this alternative activation could be performed by proteases like PR3. Contribution of IL-18 in diseases has been demonstrated in sepsis [22], arthritis [23] and inflammatory bowl disease [24] in vivo models.

The processing of the pro-inflammatory cytokines TNF-α, IL-1β and IL-18 selectively by PR3 and not by HLE points to a crucial contribution of this protease in inflammatory processes. As PR3 is the target antigen of the c-ANCA autoantibodies in Wegener's granulomatosis, a severe inflammatory autoimmune disease, the special function of this protease is of particular interest.

HLE activates EGFR

  1. Top of page
  2. Abstract
  3. Introduction
  4. Conversion of cytokines and growth factors
  5. Proteinase 3 converts TNF-α
  6. Proteinase 3 activates IL-1β
  7. Proteinase 3 activates IL-18
  8. HLE activates EGFR
  9. HLE may activate toll-like receptor-4
  10. Cathepsin G activates protease-activated receptor-4
  11. Caspase-like activity
  12. Shedding of receptors and cytokines
  13. Therapeutic approaches
  14. Conclusion
  15. Conflict of interest statement
  16. Acknowledgements
  17. References

Human leucocyte elastase has been implicated in the activation of the epidermal growth factor receptor (EGFR). HLE application on cultured keratinocytes leads to cell proliferation via EGFR involving the release of TGF-α by its proteolytic activity [25] (Fig. 4). Application of HLE on murine skin resulted in a twofold increase of epidermal layers within 3 days without a visible inflammatory infiltrate in contrast to trypsin-induced epidermal hyperproliferation [26, 27]. Trypsin is another serine protease with different cleavage preference than HLE. Concentrations of HLE used in these studies to induce proliferation of keratinocytes in vitro and in vivo are in the same range as concentrations found on the surface of psoriatic lesions [28]. The elastase activity of psoriatic lesions correlates with the level of disease and disappears after successful treatment [5]. The authors conclude that HLE is an important and relevant stimulus for epidermal proliferation in psoriasis. Using a different methodological approach it was shown that HLE leads to the induction and release of mucin in a cultured mucoepidermal cell line by proteolytical cleavage of membrane-bound TGF-α and activation of EGFR [29]. Induction of mucin by the activation of EGFR may be the major cause for hypersecretion in the lung [30]. Chronic HLE exposure to murine lungs resulted in mucus cell hyperplasia mucin induction [31]. A key role for HLE in airway secretion has been proposed for quite some time and the recent in vivo and in vitro data suggest that HLE may cause airway hypersecretion as in chronic obstructive pulmonary disease or in cystic fibrosis, where high levels of HLE are present (Fig. 3).

image

Figure 4. Activation of the g-protein coupled receptor protease-activated receptor 4 (PAR-4) by cathepsin G.

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image

Figure 3. Conversion of membrane-bound pro- TGF-α to its active soluble form by human leucocyte elastase (HLE) and consequent activation of its receptor epidermal growth factor receptor (EGFR).

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HLE may activate toll-like receptor-4

  1. Top of page
  2. Abstract
  3. Introduction
  4. Conversion of cytokines and growth factors
  5. Proteinase 3 converts TNF-α
  6. Proteinase 3 activates IL-1β
  7. Proteinase 3 activates IL-18
  8. HLE activates EGFR
  9. HLE may activate toll-like receptor-4
  10. Cathepsin G activates protease-activated receptor-4
  11. Caspase-like activity
  12. Shedding of receptors and cytokines
  13. Therapeutic approaches
  14. Conclusion
  15. Conflict of interest statement
  16. Acknowledgements
  17. References

Human leucocyte elastase upregulates IL-8 via toll-like receptor 4 (TLR-4), the LPS receptor and responsible for LPS-induced septic reactions, which became the focus of many recent studies. A TLR-4 neutralizing antibody inhibited the HLE-induced IL-8 production from cultured cells [32]. In this study HLE resulted in a decrease of TLR-4 surface expression-induced tolerance to LPS in subsequent LPS stimulations after HLE application. The same group reported previously that HLE induces IL-8 expression in bronchial epithelial cells involving the TLR signal transducers IRAK, MyD88 and TRAF-6 [33]. A possible molecular explanation was given by a study, where intraperitonally injected pancreatic elastase led to death of mice in a TLR-4 dependent manner involving the mobilization of heparan sulphate. Intraperitonally injected heparan sulphate resulted in 80% death in wild-type mice but no death in TLR-4-deficient mice [34]. Injection of pancreatic elastase resulted in 50% death in wild-type animals by its proteolytic activity and no death in TLR-4-deficient mice. The authors demonstrated that injected elastase resulted in loss of heparan sulphate from blood vessels at the injection site within 5 h of injection and conclude that activation of TLR-4 by elastase-dependent release of heparan sulphate [35, 36] might be an endogenous pathway to systemic inflammatory response syndrome-like reactions. However, this was only shown for pancreatic elastase and not for HLE.

Cathepsin G activates protease-activated receptor-4

  1. Top of page
  2. Abstract
  3. Introduction
  4. Conversion of cytokines and growth factors
  5. Proteinase 3 converts TNF-α
  6. Proteinase 3 activates IL-1β
  7. Proteinase 3 activates IL-18
  8. HLE activates EGFR
  9. HLE may activate toll-like receptor-4
  10. Cathepsin G activates protease-activated receptor-4
  11. Caspase-like activity
  12. Shedding of receptors and cytokines
  13. Therapeutic approaches
  14. Conclusion
  15. Conflict of interest statement
  16. Acknowledgements
  17. References

Besides the proteolytical cleavage of cytokines and growth receptors, the discovery of a group of seven-transmembrane receptors, which are activated directly by proteolytical cleavage and are therefore named protease-activated receptors (PARs), has dramatically changed the traditional view of protease mediated signalling. Following proteolysis, a new N-terminus of the extracellular part of PARs occurs, which now acts as a tethered ligand [37]. Four members of PARs are known. PAR-1, PAR-3 and PAR-4 are activated by thrombin, and PAR-2 by trypsin and chymotrypsin. PARs are expressed in various cells and organs and the functions described so far resemble this variety. Human neutrophil serine proteases interact with PARs. CG activates PAR-4 to initiate thrombocyte aggregation beside thrombin [38]. CG increased intracellular calcium in PAR-4-transfected fibroblasts, PAR-4-expressing oocytes and human platelets. A PAR-4 blocking antibody inhibited activation of platelets by CG and prevented calcium signalling in platelets. Thus CG may activate platelets and other cells at sites of injury and inflammation by cleaving PAR-4. PR3-cleaved peptide fragments of PAR-2 at the activation site and PR3-induced calcium response in oral epithelial cells were suppressed by prior desensitization of PAR-2, which suggested that PR3 is a PAR-2 activator [39]. Proteolytical cleavage of PARs with loss of the tethered ligand sequence resulted in an irreversible inactivation of the receptor. HLE and CG inactivate PAR-1, PAR-2 [40], and HLE also inactivated PAR-3 [41] in the same manner. To inhibit platelet aggregation potent PAR antagonists are now under clinical evaluation suggesting therapeutic potential in thrombosis, restenosis and various inflammatory disorders (Fig. 4) [42].

Caspase-like activity

  1. Top of page
  2. Abstract
  3. Introduction
  4. Conversion of cytokines and growth factors
  5. Proteinase 3 converts TNF-α
  6. Proteinase 3 activates IL-1β
  7. Proteinase 3 activates IL-18
  8. HLE activates EGFR
  9. HLE may activate toll-like receptor-4
  10. Cathepsin G activates protease-activated receptor-4
  11. Caspase-like activity
  12. Shedding of receptors and cytokines
  13. Therapeutic approaches
  14. Conclusion
  15. Conflict of interest statement
  16. Acknowledgements
  17. References

Proteinase 3 is not just active when released into the extracellular space or in a membrane bound form, but also traverses the endothelial plasma membrane and induces apoptosis [43]. The mechanism of PR3 internalization is not known, but was cell type specific as lung epithelial cells did not internalize PR3. The authors hypothesize that PR3 internalization is receptor-dependent. Exposure of endothelial cells to PR3 results in cleavage and inactivation of the transcription factor NF-kappaB and sustained activation of JNK [44]. Inhibition of caspases did not block the cleavage of p65 NF-kappaB and sequence analysis exhibited that the PR3 cleavage site was unique with respect to reported caspase cleavage sites. Recently the same group demonstrated that PR3 sidesteps caspases and cleaves the cell cycle inhibitor p21 to induce endothelial cell apoptosis. PR3 cleaves p21 at Thr80 and Gly81, a region susceptible to caspase-3 cleavage, although PR3 cleavage of p21 is caspase-independent demonstrated by using a broad-spectrum caspase inhibitor. Cleavage results in loss-of-function of p21 with nuclear exclusion and activation of apoptosis [45] (Fig. 5). The authors speculate that the kinetics of apoptotic activation would be accelerated because activation of the caspase cascade is not required for p21 cleavage. Subsequent phagocytosis of apoptotic cell would aid in the resolution of inflammation, as both the injured endothelial cells and PR3 would be removed from the site. It can be assumed that immune cells like neutrophils have evolutionarily acquired the capability to intervene into intracellular caspase cascades through released proteases like PR3 to combat foreign microbes that override normal apoptotic signals. In auto-inflammatory disorders like Wegener's disease, which is characterized by the appearance of auto-PR-3 antibodies and systemic vasculitis, PR3-induced endothelial apoptosis is an exciting new hypothesis to explain the common picture of vasculitis.

image

Figure 5. Induction of apoptosis by proteinase 3 (PR3) in endothelial cells. PR3 is internalized by a yet unknown mechanism. PR3 cleaves p21 and NF-kappaB and leads to apoptosis.

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Shedding of receptors and cytokines

  1. Top of page
  2. Abstract
  3. Introduction
  4. Conversion of cytokines and growth factors
  5. Proteinase 3 converts TNF-α
  6. Proteinase 3 activates IL-1β
  7. Proteinase 3 activates IL-18
  8. HLE activates EGFR
  9. HLE may activate toll-like receptor-4
  10. Cathepsin G activates protease-activated receptor-4
  11. Caspase-like activity
  12. Shedding of receptors and cytokines
  13. Therapeutic approaches
  14. Conclusion
  15. Conflict of interest statement
  16. Acknowledgements
  17. References

Many biological molecules like cytokines and their receptors contain putative cleavage sites for neutrophil serine proteases. It is therefore not surprising that many receptors, cytokines and other molecules have been found to be natural substrates for neutrophil serine proteases (Table 2). Cleavage of receptors of ligand-binding cytokine receptor ectodomains makes the cells insensitive to cytokines. In addition, cleaved receptors may be able to bind their ligands, which prolongs, on the one hand, their lifetime, but prevents, on the other, cytokine binding. Proteolytic cleavage of cytokines like TNF-α and IL-6 is often accompanied with a loss of function and might therefore be an important mechanism for downregulating of their inflammatory response. Considering that neutrophils themselves release some of these cytokines like IL-6 or process some of these cytokines like TNF-α and IL-1beta, the inactivation of these cytokines might represent a direct feedback mechanism. Proteolytical cleavage of cytokines does not necessarily need to result in a loss-of-function. Truncation of some chemokines like IL-8 and ENA-78 by PR3 and CG, respectively, exhibits a more active form of the chemokines. A weak point of the numerous reports is that these studies are mainly done in vitro focusing on a limited number of particular molecules. This cannot resemble the overall effect at the in vivo site. Especially, the chronology of releasing and inactivation of cytokines and their receptors might be the decisive factor of proteolytical control in inflammation. The dynamic state of activation of receptors and release of cytokines by neutrophil serine proteases, on the one hand, and inactivation of cytokines, on the other, should be considered when cytokine concentrations are analysed at sites of inflammation where high concentrations of active neutrophil proteases are present.

Table 2.  Overview of different targets of neutrophil serine proteases
TargetCleaved byHypothetical biological functionReference
Receptors
 PAR-1HLE, CGInactivation, modulation of response[40]
 PAR-2HLE, CGInactivation, modulation of response[40]
 PAR-3HLEInactivation, modulation of response[41]
 IL-2RαHLE, PR3Inhibiting cellular response and prolongation of cytokine half-life time[46]
 TNF-RIIHLEInhibiting cellular response and prolongation of cytokine half-life time[47]
 IL-6RCGInhibiting cellular response and prolongation of cytokine half-life time[46]
 C5aR (CD88)HLE, CGInhibition of chemotaxis, feedback mechanism[48]
 C3b/C4b R (CD35)HLEInhibition of complement signalling[49]
 urokinase R (CD87)HLE, CGRegulation of cell migration[50]
 GCSF-RHLEGrowth inhibition[51]
 CD43 (sialophorin)HLERegulation of adhesion[51]
 CD16 (low-affinity IgG Fc receptor)HLE [52]
 CD14HLEInhibition of LPS-mediated cell activation[53]
 CD2, CD4 and CD8CG, HLEImpairment of T lymphocytes[54]
Cytokines/chemokines
 IL-6CGRegulation of cytokine half-life time[55]
 TNF-αHLE, CGRegulation of cytokine half-life time[56]
 IL-2HLERegulation of cytokine half-life time[57]
 IL-8PR3Increased chemotaxis[58]
 ENA-78CGIncreased chemotaxis[59]
 GCSFHLEGrowth inhibition[51]
Integrins/others
 Intercellular adhesion molecule-1HLE, CGRegulation of adhesion[60, 61]
 Vascular endothelium cadherinHLE, CGRegulation of adhesion[62]
 ProepithelinHLEWound healing[63]
 TGF-β binding proteinHLE, CG, PR3Enhanced growth factor availability[64]
 IGF-binding proteinsHLE, CGEnhanced growth factor availability[65]

Therapeutic approaches

  1. Top of page
  2. Abstract
  3. Introduction
  4. Conversion of cytokines and growth factors
  5. Proteinase 3 converts TNF-α
  6. Proteinase 3 activates IL-1β
  7. Proteinase 3 activates IL-18
  8. HLE activates EGFR
  9. HLE may activate toll-like receptor-4
  10. Cathepsin G activates protease-activated receptor-4
  11. Caspase-like activity
  12. Shedding of receptors and cytokines
  13. Therapeutic approaches
  14. Conclusion
  15. Conflict of interest statement
  16. Acknowledgements
  17. References

As neutrophils are often involved in inflammatory diseases without a microbial pathogen as a cause, it is tempting to speculate that inhibition of serine proteases could be a therapeutic approach in these diseases. Indeed, animal studies showed that elafin, an inhibitor of PR3 and HLE, efficiently reduced posthypoxic damage in the lung [66], the heart [67] and a vein graft model [68]. Adenoviral augmentation of elafin protected murine lungs against acute injury mediated by activated neutrophils and bacterial infection [69]. The Kunitz-type serine protease inhibitor urinary trypsin inhibitor (UTI) has been widely used as a drug for patients with disseminated intravascular coagulation, shock and pancreatitis, especially in Japan. UTI mainly inhibits proteases, including trypsin, plasmin, CG and HLE. UTI also has anti-inflammatory properties like the downregulation of LPS-induced cytokine production in vitro. UTI protected against systemic inflammatory responses and subsequent organ injury induced by bacterial endotoxin, through the inhibition of the enhanced expression of pro-inflammatory cytokines [70]. In a recent multiple-centre, double-blind, placebo-controlled trial, the synthetic HLE inhibitor sivelestat had no effect on 28-day all-cause mortality or ventilator-free days in a heterogeneous acute lung injury patient population managed with low tidal volume mechanical ventilation [71]. Though sivelestat was effective in multiple animal models of acute lung injury [72]. A phase 3 study conducted in Japan demonstrated that sivelestat improved investigator assessment of pulmonary function improvement and significantly reduced the duration of intensive care unit stay [73]. Sivelestat as well as the specific HLE inhibitor EPI-hNE-4 had beneficial effect of bleomycin-induced lung fibrosis [74, 75].

Besides the classical administration of potential drugs new concepts of drug delivery are designed especially for the use of protease inhibitors. Nylon membranes loaded with protease inhibitors exhibited good inhibitory power with the aim of reducing the increased elastase concentration occurring during haemodialysis or extracorporeal blood circulation in patients undergoing cardiopulmonary bypass [76]. Fusion proteins consisting of protease inhibitors and target receptor ligands provide the ability to deliver antiproteases specifically, e.g. to the lumenal surface of the respiratory epithelium [77].

These are examples of new therapeutic approaches against various diseases. Future studies will have to demonstrate the therapeutic potential for selective inhibitors of neutrophil serine proteases to treat inflammatory diseases.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Conversion of cytokines and growth factors
  5. Proteinase 3 converts TNF-α
  6. Proteinase 3 activates IL-1β
  7. Proteinase 3 activates IL-18
  8. HLE activates EGFR
  9. HLE may activate toll-like receptor-4
  10. Cathepsin G activates protease-activated receptor-4
  11. Caspase-like activity
  12. Shedding of receptors and cytokines
  13. Therapeutic approaches
  14. Conclusion
  15. Conflict of interest statement
  16. Acknowledgements
  17. References

During the last 10 years evidence has pointed at other neutrophil functions than a microbial killer cell stuffed with deadly weapons and the only goal to phagocyte and eradicate the pathogen. Many studies have demonstrated that neutrophil serine proteases play an important role in cell signalling and contribute to the control of the inflammatory process. Their general processing of different local precursors to active cytokines allows a fast and specific local immune response depending on what kind and how much of the precursors are expressed. The purpose of this review was to modify the prevailing picture of serine proteases as unspecific degradation machineries by highlighting the multiple ways of cellular signalling through these enzymes.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Conversion of cytokines and growth factors
  5. Proteinase 3 converts TNF-α
  6. Proteinase 3 activates IL-1β
  7. Proteinase 3 activates IL-18
  8. HLE activates EGFR
  9. HLE may activate toll-like receptor-4
  10. Cathepsin G activates protease-activated receptor-4
  11. Caspase-like activity
  12. Shedding of receptors and cytokines
  13. Therapeutic approaches
  14. Conclusion
  15. Conflict of interest statement
  16. Acknowledgements
  17. References
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