The common denominator of several lung diseases is massive lung extracellular matrix (ECM) remodeling [
]. Lung diseases with increased lung tissue turnover include idiopathic pulmonary fibrosis (IPF) and lung cancer [
]. Protease-mediated degradation of the lung ECM is a well documented and a significant part of lung tissue degradation [
]. Many of the responsible proteases, neutrophils in particular, are associated with lung diseases such as IPF and lung cancer [
One of the signature proteins of the lungs is elastin. Elastin provides resilience and elasticity to the lungs [
]. Neutrophil elastases (NEs) are produced by neutrophils [
] and are able to degrade the otherwise stable elastin fibers in the lung [
]. NE expression and activity levels have for some time been coupled with chronic obstructive pulmonary disease (COPD). Neutrophil levels identified in the sputum of patients diagnosed with COPD have been shown to be elevated compared with controls [
]. In alignment, an increase in neutrophil cell count and cell percentage was observed in bronchoalveolar fluid (BALF) from patients with IPF compared with controls [
]. Furthermore NE may play a destructive role in lung cancer especially non-small-cell lung cancer subtypes such as squamous cell carcinoma and adenocarcinoma [
The aim of this study was to identify and quantify NE-generated elastin degradation, and assess the relationship between levels of elastin fragments and pulmonary disorders such as lung cancer and IPF that involve excessive lung tissue remodeling. We selected unique fragments of elastin degraded specifically by NE, developed a novel ELISA assay detecting exclusively NE-degraded elastin and investigated NE activity towards elastin in lung cancer and IPF.
Selection of peptides for immunizations
Decapeptides at the N- or C-terminus of known NE cleavage sites on elastin [
] were considered candidate immunogens. The decapeptides were selected for their distance to other known elastase cleavage sites in the primary structure of elastin [
]. Cleavage sites furthest away from other cleavage sites were preferred and the final number of candidates was reduced to 10. Selected decapeptide sequences were screened for theoretical cleavage sites of trypsin and chymotrypsin as well as to the cleavage sites from in-house human elastin degradome database obtained from previous study [
]. Selected decapeptides were blasted for homology to decapeptide sequences from other proteins using the NPS@: network protein sequence analysis with the UniProt/Swiss-Prot database [
]. A total of 60 mice were immunized, with groups of six mice each being immunized with a single immunogen.
The immunization [
] and fusion procedures [
] have been described elsewhere. To create the EL-NE assay immunogen, the immunogen (CGG-GGPGFGPGVV, Chinese Peptide Company, Beijing, China) was coupled to the Keyhole limpet hemocyanin (KLH) carrier protein using Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC, Thermo Scientific, Waltham, MA, USA) as the linker. The adjuvant used was Freund’s incomplete adjuvant (Sigma-Aldrich, St. Louis, MO, USA)
Characterization and selection of antibodies
Native reactivity and peptide binding of the generated monoclonal antibodies were validated by displacement of human serum in a preliminary indirect ELISA using biotinylated peptides (Biotin-KK-GGPGFGPGVV for EL-NE) on a streptavidin-coated microtitre plate (Roche, Basel, Switzerland) and the supernatant from the monoclonal hybridoma. The clones were characterized by testing the reactivity of their supernatant to the free peptide (GGPGFGPGVV for EL-NE), the elongated peptide (GGPGFGPGVVG for EL-NE) and a nonsense peptide (VGAGVPGLGV for EL-NE). The selected clones were purified using Protein G columns according to manufacturer’s instructions (GE Healthcare Life Science, Little Chalfont, Buckinghamshire, UK). All peptides were produced by the Chinese Peptide Company (Beijing, China). The final selection of the monoclonal antibody for assay development was based on high reactivity towards its free peptide and towards serum samples from patients diagnosed with IPF (ProteoGenex, Culver City, CA, USA) combined with low reactivity towards their elongated peptide and intact elastin. Peptide sequences for applied peptides were confirmed with mass spectrometry by the peptide company (Chinese Peptide Company, Beijing, China) after synthesis.
The selected monoclonal antibody was inserted into competitive ELISA systems and labeled with horseradish peroxidase (HRP) using the Lightning link HRP labeling kit according to the instructions of the manufacturer (cat. no.701-003 Innova Bioscience, Babraham, Cambridge, UK). A 96-well streptavidin plate was coated with 5 ng/mL screening peptide (Biotin-KK-GGPGFGPGVV for EL-NE) dissolved in coater buffer (10 mM PBS, 1% BSA, 0.1% Tween-20, pH 7.4, 8 g/L NaCl) and incubated for 30 minutes at 20°C. 20 μL of free peptide calibrator or sample were added in duplicate to appropriate wells, followed by 100 μL of conjugated monoclonal antibody in assay buffer (25 mM PBS, 1% BSA, 0.1% Tween-20, pH 7.4, 2 g/L NaCl) and incubated for 3 hours at 4°C. After each incubation step the plate was washed five times in washing buffer (20 mM Tris, 50 mM NaCl, pH 7.2). Finally, 100 μL tetramethylbenzinidine (TMB, Kem-En-Tec Nordic, Taastrup, Denmark) was added and the plate was incubated for 15 minutes at 20°C in the dark. All the above incubation steps included shaking at 300 rpm. The TMB reaction was stopped by adding 100 μL of stopping solution (1% H
4) and optical density was measured at 450 nm with 650 nm as the reference.
Technical validation of EL-NE
From 2-fold dilutions of human samples of serum and plasma citrate and heparin, linearity was calculated as a percentage of recovery of the undiluted neoepitope. The lower limit of detection was determined from 21 zero samples (assay buffer) and calculated as the mean + 3X standard deviation
. The lower limit of quantification was determined as the highest level of NE-generated elastin fragments with coefficient of variation (CV) below 30% reproduced in serum samples. The inter- and intra-assay variation was determined by 10 independent runs of 8 samples that covered the detection range of the EL-NE. Besides five human serum samples, the 8 samples included one bovine serum sample, one sample with the free peptide in human serum and one sample with the free peptide in buffer. The freeze-thaw recovery of human serum and citrate and heparin plasma was determined by measuring the NE-degraded levels of elastin in three samples of each, which were exposed to four freeze-thaw cycles and compared to NE-generated levels of elastin prior to the first cycle. Analyte stability was determined by the levels of NE-degraded elastin in three samples each of human serum and plasma citrate and heparin after either 4°C or 20°C storage for 24 hours and compared with the levels at zero hours.
The reactivity of the EL-NE antibody towards the free peptide (GGPGFGPGVV) was compared with its reactivity to the elongated peptide (GGPGFGPGVVG), a nonsense peptide (VGAGVPGLGV) as well as to the free peptide where a nonsense peptide was applied as screening peptide (VGAGVPGLGV-KK-Biotin). The added peptide doses were 119 nM, 59 nM, 30 nM, 15 nM, 7 nM, 4 nM, 2nM and 0 nM.
Levels of NE-degraded elastin were determined in the presence of elastin
in vitro cleaved with: matrix metalloproteinase (MMP)-2, MMP-7, MMP-9, MMP-12 or NE, NE in NE buffer as well as intact elastin dissolved in NE buffer (all incubated for 48 hours at 37°C). Elastin was incubated once with each enzyme. Enzyme:protein ratios were 1:100 (MMPs) or 1:200 (NE) (weight/weight). For cross-reactivity towards CatG cleavage, enzyme:protein ratios were 1:50 (NE) and 1:15 (CatG) (weight/weight). Incubation times for the cleavages, intact elastin, NE and CatG were 24 hours at 37°C. Activity tests were performed on proteases prior to cleavage. All material was diluted 100x in assay buffer before measurement. Insoluble elastin was purchased from Sigma-Aldrich (cat. no. E6777, St. Louis, MO, USA); MMP-2 and MMP-9 from Calbiochem (cat. no. 444213 and 444231, Whitehouse Station, NJ, USA), MMP-7 and MMP-12 from R&D Systems (cat. no. 907-MP-010 and 917-MP-010, Minneapolis, MN, USA), cathepsin G from Elastin Product Company (cat. No. SG623, Owensville, MO, USA) and NE from Abcam (cat. no. ab80475, Cambridge, UK).
Clinical validation of EL-NE
Levels of NE-degraded elastin were determined in serum from patients diagnosed with IPF (n = 10, mean age 74 years, 20% female) and compared with healthy age- and sex-matched controls (n = 9, mean age 72 years, 22% female). NE-generated elastin levels were also measured in serum from patients diagnosed with lung cancer (n = 40, of which n = 16 had squamous cell carcinoma, n = 16 had adenocarcinoma, n = 8 had small cell lung cancer; mean age 59 years, 25% female) and compared with healthy age- and sex-matched controls (n = 12, mean age 60 years, 25% female). All controls were derived from a previously described study [
]. Patient samples were obtained from the commercial vendor Proteogenex (Culver City, CA). After signed consent from patients and approval by the appropriate Institutional Review Board or Independent Ethical Committee, serum had been collected from patients with IPF or lung cancer. According to Danish law, it is not required to obtain ethical approval when measuring biochemical markers in previously collected samples; hence, there was no additional ethical approval for this study. Samples were all collected, processed, and stored in a similar fashion until analyzed. Patient samples were collected prior to surgery. Additional patient demographics and clinical information is presented in table S1 (see Additional file
: Appendix 1).
The geometric means (95% CI) of serum levels of NE-generated elastin fragments in diagnosed patients were compared with their respective controls using the two-sided non-parametric Mann Whitney test. All statistical analyses were performed in MedCalc from MedCalc Software (Ostend, Belgium). Results were considered statistically significant if p < 0.05.
Detailed materials and methods for the EL-NE-B competitive ELISA (VGAGVPGLGV) can be seen in Additional file
: Appendix 1.
To our knowledge this is the first quantification of an NE-generated elastin fragment in human serum. The main findings of this study were: 1) the selection and quantification of a unique NE-generated elastin fragment; 2) development of the EL-NE antibody which was specific towards a NE cleavage site on elastin and not towards other proteases; 3) development of a competitive ELISA assay for the assessment of NE-degraded elastin in serum; 4) demonstration that levels of NE-degraded elastin were significantly elevated in serum from IPF and lung cancer patients compared with healthy controls.
Selection of the EL-NE assay
The antibody for the EL-NE assay was selected from a total of 60 antibodies. Besides that used in the EL-NE assay, other promising antibodies were developed towards NE-specific degraded elastin. As an example EL-NE-B (see Additional file
) demonstrated high specificity towards its free peptide and elastin cleaved with NE. However the clinical relevance of EL-NE-B was low as it could not discriminate between IPF and healthy controls. Furthermore, using the EL-NE-B assay, NE-degraded elastin was only detectable in very low levels in serum from healthy and diseased patients.
Technical validation and specificity of EL-NE
The low inhibition by the elongated peptide demonstrated that the antibody only recognizes the neoepitope occurring after proteolytic cleavage between Val334 and Gly335. The high levels of the EL-NE fragment in elastin cleaved
in vitro with NE, compared with MMPs or Cat-G, demonstrated that the EL-NE assay is specific for NE-degraded elastin and that the EL-NE fragment is not a total protein marker since reactivity with intact elastin was minimal. The selected NE cleavage site on elastin is not adjacent to common proteases such as trypsin or chymotrypsin. The levels of the elastin fragment could be recovered after four freeze/thaw cycles and 24 hours storage and the NE-degraded elastin levels decreased in proportion to the number of times samples were diluted. Thus we developed an assay that quantifies a specific NE-derived elastin fragment.
When quantifying the effectiveness of NE inhibitors in pulmonary studies the determination of NE activity often includes the isolation of neutrophils, mainly from the blood [
]. After addition of substrates, such as methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide the NE activity can be quantified. The development and production of NE substrates are considerably faster than monoclonal antibodies. However, the EL-NE assay does not depend on cell extraction and can be applied directly to serum samples. Cell and protease extraction is a significant source of variation and the process may affect NE activity. As demonstrated in this study, the EL-NE assay may recover the majority of analytes after a series of freeze/thaw cycles and storage above freezing temperatures. It is questionable whether ECM proteases stored in serum could maintain the majority of their activity during freeze/thaw cycles.
Neutrophil elastase activity in IPF
The increased levels of NE-degraded elastin in IPF patient samples indicated that NE-specific degradation of elastin may have a relevant diagnostic role within IPF. In IPF the role of MMPs has received much focus [
] but NE activity in IPF can also be an indicator of survival rate [
]. The EL-NE IPF data corresponds well with the discovery that IPF patients with a low BALF neutrophil cell percentage were more likely to survive than IPF patients with a higher percentage [
]. The increase in neutrophils in IPF patients may explain the higher levels of circulating elastin fragments in IPF patients compared with controls. EL-NE fragment levels of 2 nM in the controls may be caused by background ECM remodeling combined with matrix effects from the serum. Further studies with control and patient samples obtained with identical standard operating procedures are needed to investigate elastin fragment levels in healthy controls. As the formation of elastin mostly occurs during the younger years increased levels of NE-degraded elastin fragments may be an indicator of imbalanced tissue remodeling, a significant contributor to IPF, in adults. Larger longitudinal studies may reveal the prognostic potential for the EL-NE assay in IPF.
NE activity in lung cancer
Elevated levels of NE have been coupled with cancer invasion and metastasis in lung cancer [
] and most likely explains the increased levels of NE-generated elastin fragments in lung cancer patients. High NE proteolytic activity on elastin may therefore be related to the excessive ECM turnover observed during the progression of lung cancer.
All clinical samples were collected in cross-sectional studies. Longitudinal studies, with several time points and a larger representation of varying disease severity would enable a better description of NE-degraded elastin as a marker of excessive ECM lung turnover. Controls were not matched according to race or smoking history and were collected at different centers than the disease samples. The clinical validation of EL-NE was performed in serum. Future studies should include BALF and plasma samples.
In conclusion we have utilized specific NE-elastin cleavage sites to develop a non-invasive ELISA assay (EL-NE) to assess levels of NE-generated elastin fragments. The EL-NE analyte can be recovered after dilution, freeze/thaw cycles and temporary storage above freezing temperature. The developed EL-NE assay was specific towards elastin cleaved
in vitro with NE. We conclude that the quantification of the EL-NE analyte may be a marker of the increased tissue remodeling observed in pulmonary disorders such as IPF and lung cancer.
JHK, MAK and DJOL have filed the patent 1400472.5 'Elastin lung disease diagnostics'. The remaining authors declare that they have no competing interests.
JHK contributed to the design of the immunoassay and performed all laboratory procedures, calculated the results, performed the statistical analysis and drafted the manuscript; MK contributed to the design of the immunoassay, data interpretation, manuscript draft and manuscript revision; JMBS contributed to statistical analysis, data interpretation and manuscript revision; NWI contributed to data interpretation and manuscript revision; CD contributed to data analysis and manuscript revision; BS contributed to manuscript revision, PH contributed to manuscript revision; DJL contributed to the design of the immunoassay; data interpretation, manuscript draft and manuscript revision. All authors read and approved the final manuscript.