Abstract
Objective. Neutrophil elastase is secreted by neutrophils during activation and circulates in the plasma where it can play a role in inflammation and fibrosis. This study examines the role of neutrophil elastase in SSc, a systemic CTD that is typified by vascular dysfunction, tissue fibrosis and inflammation.
Methods. Serum neutrophil elastase and α1-anti-trypsin concentrations were assessed in SSc patients and healthy controls by ELISA. Serum neutrophil elastase activity was assessed by the elastase-dependent conversion of methoxy-succinyl-alanyl-alanyl-prolyl-valyl-p-nitroanilide to p-nitroanilide using a colourimetric assay. Elastase concentration and activity were correlated with clinical disease features.
Results. Serum neutrophil elastase concentration and activity were equivalent in patients and controls; however, in SSc serum, there was an increase in elastase activity for each unit of elastase concentration (P = 0.03). This was due to a decrease in serum α1-anti-trypsin concentrations (P = 0.04). Serum elastase concentration (P = 0.03) and activity (P = 0.02) were significantly lower in RNP-positive patients and serum elastase concentrations were lower in ANA-positive patients (P = 0.003).
Conclusions. Relative deficiency in serum α1-anti-trypsin concentrations in SSc could have important and pathogenically relevant effects since elastase has pro-inflammatory and pro-fibrotic roles. Elastase inhibitors are available in clinical practice and could represent potential therapeutic options in SSc.
Keywords: Neutrophil elastase, α1-Anti-trypsin, Systemic sclerosis, Serine protease, Innate immunity, Neutrophil
Introduction
Neutrophil elastase is a serine protease that is stored in the azurophilic granules of neutrophils. It predominantly functions as an intracellular anti-microbial protein and is released into the phagolysosome following phagocytosis to mediate bacterial digestion. However, in addition, it is secreted by the cell. In the extracellular space, neutrophil elastase has additional functions in the regulation of inflammation and it is implicated in inflammatory and fibrotic conditions, including fibrotic lung disease [1, 2]. Elevations in serum elastase have previously been reported in patients with SSc where it was associated with lung disease [3].
Serum elastase activity is regulated by serine protease inhibitors. The main intracellular inhibitor of elastase is serpin peptidase inhibitor clade B member 1 (SERPIN B1), whereas the main extracellular inhibitor of neutrophil elastase is α1-anti-trypsin and additional inhibition is mediated by α2-macroglobulin, elafin and secreted leucocyte proteinase inhibitor (SLPI) [4]. Neutrophil elastase can also be expressed on the neutrophil membrane, where it occupies low- and high-affinity binding sites. Membrane expression is increased by neutrophil activation with cytokines such as TNF-α and IL-8 [5]. The increase in membrane expression is more significant than the amount secreted extracellularly during typical activation. Plasma membrane-bound elastase has the same catalytic functions as soluble elastase, but there is some evidence that the membrane-bound enzyme is relatively resistant to α1-anti-trypsin [6, 7].
Neutrophils were shown by Hussein et al. to be increased in lesional biopsies of SSc patients compared with controls [8]. Others have explored neutrophil function in SSc, in particular their ability to contribute to oxidative stress by the production of reactive oxygen species. The data are contradictory and largely limited by old-fashioned neutrophil isolation procedures that can lead to neutrophil activation [9, 10]. A recent study has, however, shown that neutrophils produce less reactive oxygen species in vitro compared with control neutrophils when unstimulated [11]. In agreement with this we have found that neutrophils from patients with SSc are hypofunctional in tests of reactive oxygen species generation and chemotaxis (unpublished data). This may reflect in vivo stimulation, and hence in vitro exhaustion. Proteomic studies show that SSc neutrophils have increased expression of proteins that are also increased on stimulation with lipopolysaccharide or TNF, again indicative of neutrophil activation in vivo (unpublished data).
Since neutrophil elastase has established roles in other inflammatory and fibrotic disorders, we hypothesized that neutrophil elastase could be an important mediator in the pathogenesis of SSc. To explore this hypothesis, the concentration and catalytic activity of neutrophil elastase in SSc serum was compared with controls. In addition, the membrane expression of elastase was measured in SSc neutrophils compared with controls, and clinical correlates were studied.
Methods
The study was approved by the Sefton Local Research Ethics Committee, in accordance with the Declaration of Helsinki. Informed written consent was taken from patients with SSc [12] and from healthy volunteers. Thirty millilitres of heparinized venous blood were taken from the subjects. Peripheral blood was separated into neutrophil and mononuclear cell fractions using Polymorphprep (as described in the manufacturer’s instructions). Contaminating erythrocytes were removed using ammonium chloride lysis buffer (KHCO3 3.4 mM, NH4Cl 155 mM and EDTA 96.7 µM). Neutrophils were routinely examined for purity using morphological analysis of cytospins after staining with Rapid Romanowsky: purity was >95% immediately after isolation. Neutrophils were resuspended in Roswell Park memorial institute (RPMI) 1640 + 25 mM 4-(2-hydroxyethyl)-1-piperazinethanesulfonic acid (HEPES) + 2 mM glutamine at a concentration of 5 × 106 cells/ml.
Serum elastase concentrations
Neutrophil elastase and α1-anti-trypsin levels were measured in SSc patient and control serum using a PMN elastase ELISA (Bender, Vienna) and an α1-anti-trypsin ELISA (Abnova, Taipei), respectively, according to the manufacturer’s instructions. The intra- and interassay coefficients of variation for the PMN elastase ELISA were 4.8 and 5.6%, respectively, with standard range 0.16–10 ng/ml and sensitivity 1.98 ng/ml. The epitope for the PMN elastase ELISA is unknown and it is unknown whether both active and inactive enzymes are detected using this assay. The intra- and interassay coefficients of variation for the α1-anti-trypsin ELISA were 4.2 and 7.1%, respectively, with standard range 0.039–40 µg/ml and sensitivity 20 ng/ml.
Neutrophil elastase concentrations in neutrophil culture supernatants
Neutrophils were isolated from SSc and healthy control blood, and were cultured for 6 h with gentle agitation at 37°C at a density of 107 cells/ml in RPMI 1640 + 25 mM HEPES + 2 mM glutamine. Cells were precipitated by centrifugation (1000 g for 5 min) and the neutrophil elastase concentration in the supernatants was measured by ELISA (Bender, Vienna).
Serum neutrophil elastase enzymatic activity
Following experiments to optimize substrate concentration and length of incubation, serum elastase activity was measured using a colourimetric assay. In 96-well clear plastic plates, 150 µl of elastase buffer (0.1 M HEPES, 0.5 M NaCl, pH 7.5) was added to 50-µl serum samples from patients with SSc and healthy controls. The substrate (15 mM methoxy-succinyl-alanyl-alanyl-prolyl-valyl-p-nitroanilide) was added to a final concentration of 750 µM. The reaction was incubated in the dark at 37°C for 6 h. The elastase-dependent conversion of colourless methoxy-succinyl-alanyl-alanyl-prolyl-valyl-p-nitroanilide to yellow p-nitroanilide was measured as a change of absorbance at 405 nm on a plate reader. Standards of known concentration were used to generate a calibration curve (data not shown). The intra- and interassay co-efficients of variation were 1.2 and 6.8%, respectively.
Clinical data
Clinical data were collected on all patients as a part of routine clinical care and included Rodnan skin score, neutrophil count, autoantibody profile, medications, major organ involvement and disease duration (defined as time since onset of first non-Raynaud’s symptom). Pulmonary artery hypertension was confirmed by right heart catheterization. Pulmonary fibrosis was diagnosed according by pulmonary function tests and high resolution computed tomography (HRCT), subdivisions for severity were not applied.
Neutrophil membrane elastase expression
Neutrophil membrane elastase expression was measured by flow cytometry. Neutrophils were isolated from SSc and healthy control blood as previously described. Cells were resuspended in 200 µl of phosphate buffered saline (PBS) 2% BSA, and incubated for 30 min in the dark at 4°C with 2 µl elastase monoclonal antibody raised in rabbit (Calbiochem), following experiments to find the optimal antibody dilution (data not shown). The cells were washed three times in PBS and resuspended in PBS 2% BSA and 2 µl FITC-conjugated anti-rabbit IgG secondary antibody. Cells were incubated for 30 min at 4°C in the dark. Cells were washed three times in PBS, resuspended in PBS and analysed on the flow cytometer on the FITC channel. Mean fluorescence readings were corrected to a secondary antibody control.
Statistical methods
The data were non-normally distributed and were therefore compared using the Mann–Whitney U-test.
Results
Clinical characteristics
Table 1 shows the clinical characteristics of the SSc patients.
Table 1.
Patient characteristics
| Clinical feature | Median Interquartile range |
|---|---|
| Disease duration, months | 40 (21–96) |
| Neutrophil count × 109/l | 4 (2.4–4.8) |
| Rodnan skin score | 6 (3–10) |
| n/N (%) | |
|---|---|
| Limited SSc | 29/33 (88) |
| Diffuse SSc | 4/33 (12) |
| ANA | 26/31 (84) |
| Anti-centromere | 13/31 (42) |
| Anti-RNP | 9/31 (29) |
| Anti-Scl70 | 9/31 (29) |
| Lung involvement | 10/33 (30) |
| Pulmonary artery hypertension | 5/33 (15) |
| DMARDs | 14/33 (42) |
| HCQ | 3/33 (9) |
| MMF | 4/33 (12) |
| MTX | 1/33 (3) |
| CYC | 1/33 (3) |
| PRED | 3/33 (9) |
| AZA | 1/33 (3) |
| Bosentan | 1/33 (3) |
| Sildenafil | 3/33 (9) |
PRED: prednisolone.
Serum neutrophil elastase levels were equivalent in patients with SSc compared with controls
There was no significant difference between SSc patient and control serum levels of neutrophil elastase (P = 0.11) (Fig. 1A).
Fig. 1.
Elastase and α1-anti-trypsin were measured in SSc and control serum by ELISA, serum elastase activity was measured by the elastase-dependent conversion of methoxy-succinyl-alanyl-alanyl-prolyl-valyl-p-nitroanilide to p-nitroanilide. No difference was found in serum elastase concentration (A) or activity (B). There was a significant decrease in the ratio of elastase concentration : elastase activity in SSc serum compared with controls (C): this was due to a decrease in serum α1-anti-trypsin concentration in SSc serum compared with controls (D).
Neutrophil elastase concentrations in control and SSc neutrophil culture supernatants are equivalent
There was no difference in the levels of neutrophil elastase in supernatants following a 6-h culture of control and SSc neutrophils in RPMI 1640 + 25 mM HEPES + 2 mM glutamine (P = 0.53).
Serum neutrophil elastase activity is equivalent in SSc patients and controls
The serum neutrophil elastase activity was measured in SSc patients and controls by the elastase-dependent conversion of methoxy-succinyl-alanyl-alanyl-prolyl-valyl-p-nitroanilide to p-nitroanilide. No difference in serum elastase activity was found between SSc patients and controls (P = 0.91) (Fig. 1B). However, there was a proportion of patients [6/18 (33%)] that had high serum elastase activity (>0.15 U/ml) compared with 1/9 (11%) controls. These patients had either early disease <36 months or a higher Rodnan skin score of >9. There was a significant decrease in the ratio of serum elastase concentration: activity (P = 0.03) (Fig. 1C). There were no distinguishing clinical features in the patients with a low serum elastase concentration:activity ratio (<50 000).
Clinical correlations with serum neutrophil elastase concentration and activity
Disease subtype, disease duration, major organ involvement, neutrophil count or DMARD use did not correlate with either serum neutrophil elastase concentration or activity. Serum elastase concentration was significantly lower in RNP and ANA-positive patients compared with antibody-negative patients (P = 0.03 and P = 0.003, respectively). In addition, serum elastase activity was significantly lower in RNP antibody-positive patients (P = 0.02).
Membrane expression of neutrophil elastase
No difference was found in the membrane expression of neutrophil elastase between SSc and control neutrophils.
Serum α1-anti-trypsin
Serum α1-anti-trypsin concentrations were significantly lower in SSc patients compared with controls (P = 0.04, n = 20) (Fig. 1D).
Discussion
In this study, we identified no difference in serum elastase concentration or catalytic activity in SSc patients compared with controls. This contradicts a previous study by Hara et al. [3], which observed an increase in serum elastase concentration in both limited and diffuse SSc patients. However, an examination of their data reveals that the serum elastase levels measured in this study were similar in magnitude and variation to their observations in SSc patients. In this study, however, higher serum elastase concentrations and greater variance in concentration were found in the control cohort, whereas the previous study showed consistently low levels in all controls. The previous study did not examine elastase activity.
Hara et al. [3] also reported that serum elastase levels were more likely to be outside the normal range in patients with joint involvement, and they observed that most patients who were ACA positive were likely to have normal levels of elastase. We did not record joint involvement as a clinical outcome in our cohort. It is interesting to note, however, that we observed lower serum elastase levels in RNP-positive patients, since these patients would be expected to have higher rates of joint involvement. Hara et al. [3], did not record RNP antibody status in their study. It would be interesting to correlate the data with anti-RNA polymerase III expression as this antibody can be associated with inflammatory skin disease; however, we do not routinely perform this autoantibody on our patients.
It may seem somewhat surprising that Hara et al. [3] described such an increase in serum elastase levels, since only 2% of serum elastase is released during neutrophil activation and raised serum levels are usually only found in situations where there is pronounced neutrophil infiltration, frustrated phagocytosis or excess neutrophil apoptosis, which overwhelms phagocytic clearance [5, 13, 14]. As none of these is implicated in SSc, we would not expect to find elevated serum levels, an expectation confirmed in our studies. Certainly, direct observations do not show significant neutrophilic infiltration and there is no evidence of excessive neutrophil apoptosis.
CRP at the time the assays were collected was normal in all but five patients. The mean CRP was <5 (where 5 is the lower limit of detection in the assay used). Of the five patients with elevated CRP, none of these had an elevated serum elastase nor were they patients with a low serum α1-anti-trypsin. One of these patients, with a CRP of 8, did have a low serum elastase concentration:activity ratio (<50 000); however, the others (CRP 8–19) did not.
We did not observe any increase in the membrane expression of elastase on SSc neutrophils. Secretion of azurophilic granule contents during degranulation or activation by cytokines results in the increased expression of neutrophil elastase at the membrane. Therefore, this observation argues against significant degranulation of the azurophilic granules in SSc neutrophils [5].
We did observe a decrease in the ratio of elastase concentration : activity in SSc serum. This may imply a decrease in the serum concentration or activity of neutrophil elastase inhibitors. This was confirmed by a decrease in serum α1-anti-trypsin concentrations in SSc patients, which is the main inhibitor of elastase function in the serum. In addition, serum elastase inhibitors can be oxidized in the presence of ROS and this decreases their affinity for elastase, reducing their inhibitory capacity [15, 16]. There is evidence for increased oxidative stress in SSc [17].
The stoichiometry of the interaction between α1-anti-trypsin is 1 : 1 and the inhibition of elastase is linear for molar ratios of α1-anti-trypsin : elastase of up to 2. Simultaneous measurements of α1-anti-trypsin and elastase were not taken in this study, and therefore the molar ratios in these patients cannot be derived. However, in this disease, it is unlikely to be the serum molar ratios that are relevant rather the local tissue levels. Tissue levels of α1-anti-trypsin are likely to be dictated by the serum concentration since it is produced by the liver and not produced distally [4]; however, the molar concentration of elastase is likely to be dictated locally where, for instance, interaction of neutrophils with activated endothelium and pro-inflammatory cytokines may lead to elastase release.
Neutrophil elastase has pleiotropic roles in the extracellular environment including profound effects on inflammation [1, 2]. Neutrophil elastase activates and degrades inflammatory cytokines, regulates neutrophil attachment, activates proteinase-activated receptor 2 (PAR-2) leading to the release of IL-8 and macrophage chemotactic factor-1, activates toll-like receptor-4 leading to the release of IL-8, cathepsin B and MMP-2. Elastase can cleave TNF p75 receptor and IL-6R from cells leading to an increased pool of soluble receptor. This in turn leads to inactivation of soluble TNF-α but increased IL-6 trans-signaling. Elastase can cleave the phosphatidylserine receptor, leading to defects in the phagocytic clearance of apoptotic cells and can enter endothelial cells, where it cleaves nuclear factor kappa B leading to endothelial cell apoptosis.
Although the role of neutrophil elastase in fibrosis is well established, the mechanism remains unclear. Certainly, elastase-deficient mice are resistant to bleomycin-induced fibrosis and treatment with elastase inhibitors also abolished fibrosis in this model [18–21]. Neutrophil elastase can cleave TGF-β-binding protein leading to release of latent TGF-β from extracellular matrix stores [22]. It has also been shown to release PDGF and VEGF from stores by a similar mechanism [23]. All of these cytokines are implicated in the pathogenesis of SSc; TGF-β and PDGF are pro-fibrotic and VEGF is pro-angiogenic. Neutrophil elastase has other roles in promoting the activity of TGF-β. Intra-tracheal instillation of elastase in mice leads to a time-dependent increase in the TGF-β content of the bronchoalveolar lavage fluid [24]. In elastase-deficient mice, the resistance to bleomycin is associated with an inability to activate TGF-β [18].
It is interesting that serum elastase concentrations and activity are lower in RNP-positive patients. This may imply that there is a different pathological process involved in RNP-positive patients and that neutrophil elastase is unlikely to be a significant mediator in these patients. In fact, serum elastase concentrations were lower in RNP-positive patients than controls (P = 0.008). This may represent an accelerated loss of neutrophil elastase in RNP-positive patients.
Analysis of the literature shows that neutrophil elastase could be an attractive mediator in SSc. It can promote chronic inflammation and TGF-β-dependent fibrosis, and cause endothelial cell apoptosis. Serum deficiency in elastase inhibitors could lead to a localized excess of elastase activity despite normal serum concentrations. This could have potential therapeutic implications as neutrophil elastase inhibitors already exist in clinical practice and could be used in SSc patients.
Acknowledgements
We would like to thank Steph Ling for the donation of samples and Jenny Hawkes for technical assistance.
Funding: This work was supported by the Medical Research Council, UK [G0600404].
Disclosure statement: The authors have declared no conflicts of interest.
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