Introduction
A novel feature of the biology of polymorphonuclear granulocytes (PMNs) is their ability to generate neutrophil extracellular traps (NETs)
[
1] via a distinct process of cell death termed NETosis
[
2]. NETs consist of extruded chromosomal DNA decorated with granular components that include antimicrobial peptides and proteases. The molecular pathways leading to NETosis encompass calcium mobilization, generation of reactive oxygen species (ROS), nuclear delobulation involving the enzymatic activities of myeloperoxidase (MPO) and neutrophil elastase (NE), and chromatin modification via the citrullination of histones by peptidyl arginine deiminase 4 (PAD4)
[
2‐
6].
A number of studies have implicated NETs in the etiology of autoinflammatory or autoimmune conditions such as preeclampsia, Felty syndrome, systemic lupus erythematosus (SLE), multiple sclerosis, and, most recently, rheumatoid arthritis (RA)
[
7‐
13]. In the context of RA, these findings are especially interesting, as NETs have been proposed to contribute to the generation of anti-citrullinated protein antibody (ACPA) autoantigens, and may also be a target for autoantibodies
[
13,
14]. PMNs isolated from RA patients showed an increased propensity to undergo spontaneous and LPS-induced NETosis, which was in part mediated by TNF and IL-17 and could be inhibited by blocking NADPH oxidase or PAD4. Whereas the citrullinated autoantigens vimentin and α-enolase were expressed on NETs from RA PMNs, antibodies to the former were able to induce NET formation by healthy control PMNs
[
13,
14].
As we had previously detected significantly increased concentrations of cell-free DNA in the sera of RA patients compared with healthy controls, we were intrigued whether the provenance of this material involved NETosis
[
15]. The premise for the current investigation was that a link between circulating cell-free DNA levels and NETs has previously been made in a number of conditions, including preeclampsia, sepsis, cancer, thrombosis, or even storage of blood-transfusion products
[
16‐
19].
In view of these findings and reports on the complex involvement of neutrophil NETs in autoimmunity, we sought to investigate the NETotic response of PMNs in RA, with particular regard to the underlying signal-transduction cascade, and whether the products of overt NETosis could be diagnostically useful.
Materials and methods
Human subjects
All patients fulfilled the American College of Rheumatology classification criteria for RA, or for systemic lupus erythematosus, respectively. Healthy volunteers, matched for gender and age, were recruited at the hospitals or at the Blood Bank of the Swiss Red Cross, Basel. Inclusion criteria for healthy controls were fair general condition, age ≥28 and ≤70 years, and for blood donors fulfilling national criteria for blood donation. Exclusion criteria were current or previous systemic autoimmune disease, asthma and reconvalescence after major illness, surgery, current medication with corticosteroids, immunosuppressive agents, and malignant neoplasia or chemotherapy within 5 years before recruitment for the study. RA cases had a DAS ≥2.6, were from age ≥27 to ≤70 years, and had no other systemic autoimmune disease, including ankylosing spondylitis and psoriatic arthritis.
Exclusion criteria were corticosteroids ≥40 mg equivalent of prednisone daily, and those mentioned earlier for healthy controls. Informed, written consent was obtained from all subjects in the study, which was approved by the Cantonal Ethical Review Boards of Aargau-Solothurn and Basel/Basel-Land, Switzerland.
Preparation of plasma and serum
Plasma and serum were collected and processed as described previously
[
15]. Samples were studied immediately or stored at -80°C until analysis.
Cell isolation
PMNs were isolated with Dextran-Ficoll density centrifugation
[
8]. Cell viability was 96% to 98%, with a purity of >95% PMNs. Neutrophils seeded in 24-well plates were allowed to settle for 1 hour at 37°C under 5% CO
2 before further experimentation.
Cell-free DNA isolation and quantification
Cell-free DNA extracted from 850 μl plasma or serum by using the QIAamp Circulating Nucleic Acid Kit (Qiagen, Valencia, CA, USA) was quantified by TaqMan real-time PCR (StepOne Plus Real-Time PCR System, Applied Biosystems, Foster City, CA, USA) specific for the glyceraldehyde-3-phosphate dehydrogenase (
GAPDH) gene
[
15].
Detection of neutrophil elastase (NE), myeloperoxidase (MPO), and cell-free nucleosomes
The concentrations of neutrophil elastase (NE) and myeloperoxidase (MPO) were measured with sandwich ELISA (Elastase/a1-PI Complex ELISA Kit, Calbiochem/EMD, Gibbstown, NJ, USA) and the human (MPO ELISA Kit, Hycult Biotech, Plymouth Meeting, PA, USA) respectively. Nucleosomes were measured by using the Human Cell Death Detection ELISA
PLUS (Roche Diagnostics, Basel, Switzerland). Cell-culture supernatants were incubated with DNaseI (10 U for 5 minutes) (Roche Diagnostics) before analysis
[
20].
MPO/DNA complex detection
MPO is present on extruded NETs. To detect such structures, NET-associated MPO/DNA complexes were quantified by using a modified capture ELISA
[
21]. In brief, NET-associated MPO in serum or culture supernatant was captured by using the coated 96-well plate of the human MPO ELISA Kit, (Hycult Biotech), after which the NET-associated DNA backbone was detected by using the detection antibody of the Human Cell Death Detection ELISA
PLUS (Roche Diagnostics).
PAD4/DNA-complex detection
To detect the presence of PAD4 on extruded NETs in culture supernatants after spontaneous NETosis, cell-free PAD4/DNA complexes were quantified by using a modified capture ELISA, akin to that described for MPO earlier. In brief, cell-free PAD4 was captured by using the coated 96-well plate of a commercial human PAD4 ELISA (USCN Life Science, Inc., Wuhan, China), and associated DNA was detected by using Human Cell Death Detection ELISAPLUS kit (Roche Diagnostics).
ROS generation analysis
ROS was measured by using a 2′,7′-dichloro dihydrofluorescein diacetate (DCFH-DA) assay
[
22]. The 5 × 10
5 cells in a final volume of 500 μl were incubated for 30 minutes with 25 μ
M DCFH-DA (Sigma-Aldrich, St. Louis, MO, USA). Fluorescence was measured with flow cytometry (FACSCalibur; BD Biosciences, San Jose, CA, USA).
Fluorescence and scanning electron microscopy
The 5 × 10
4 cells isolated PMNs seeded on poly-L-lysine-coated coverslips (BD Biosciences) were stimulated with phorbol-12-myristate-13-acetate (PMA, Sigma-Aldrich) for 90 minutes and dehydrated with a graded ethanol series (30%, 50%, 70%, 100%)
[
8], coated with 2-nm platinum, and analyzed with a Nova NanoSEM 230 scanning electron microscope (FEI Co., Hillsboro, OR, USA). PMNs were incubated for 10 minutes with 5 μ
M Sytox Green dye (Invitrogen Life Technologies, San Diego, CA, USA) for assessment of NETs with an Axiovert fluorescence microscope (Carl Zeiss) coupled to a Zeiss AxioCam color CCD camera (Carl Zeiss Microimaging, Oberkoch, Germany)
[
8,
23].
Immunohistochemical staining and quantification of NETs
The 5 × 104 isolated PMNs were seeded on poly-L-lysine-coated glass coverslips (BD Biosciences, San Jose, CA, USA) in tissue-culture wells and allowed to settle before stimulation, as described earlier. Coverslips were rinsed with ice-cold HBSS and the cells fixed with 4% paraformaldehyde and blocked overnight (HBSS with 10% goat serum, 1% BSA, 0.1% Tween20, and 2 mM EDTA) at 4°C. NETs were detected with rabbit anti-NE (Abcam, Cambridge, MA, USA), rabbit anti-MPO (Dako, Glostrup, Denmark), two different rabbit anti-PAD 4 (Abcam), mouse anti-PAD4 (Abcam), mouse anti-histone H1 + core proteins (EMD Millipore, Billerica, MA, USA), and rabbit anti-citrullinated histone H3 (citH3, Abcam). Secondary antibodies were goat anti-rabbit IgG AF555, goat anti-rabbit IgG AF488 (Invitrogen Life Technologies, San Diego, CA, USA), and goat anti-mouse IgG AF647. DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich), and NETs were visualized by using a Zeiss Axioplan 2 Imaging fluorescence microscope in conjunction with a Zeiss AxioCam MRm monochromatic CCD camera and analyzed with Axiovision 4.8.2 software (Carl Zeiss). A minimum of 20 fields (at least 1,000 PMNs) per case was evaluated for MPO/NE and DNA co-staining; nuclear phenotypes and NETs were counted and expressed as percentage of the total number of cells in the fields.
RA serum depletion, IgG purification, and quantification of NETs
After three washes with PBS, 200 μl protein G agarose (Pierce Biotechnology Inc, Rockford, IL, USA) was incubated with 200 μl ACPA + and ACPA- RA or control serum diluted in an equal volume of phenol red-free RPMI 1640 medium overnight at 4°C. The serum/protein G agarose mixture was centrifuged at 2,500 g for 5 minutes, and the supernatant (IgG-depleted serum) was carefully transferred into a new Eppendorf microcentrifuge tube. The protein G agarose pellet was gently washed 3 times with 500 μl ddH2O, and the bound antibody was released by the addition of 50 μl 0.1 M glycine pH 2–3, and immediately equilibrated with 10 μl of 1 M Tris pH 7.5-9. All protein concentrations were determined with the MN Protein Quantification Assay (Macherey-Nagel GmbH, Düren, Germany), and isolation of IgG was verified with Coomassie staining of SDS-PAGE.
Neutrophils from healthy donors (n = 3) were isolated and cultured for 2 hours in 96-well culture dishes (Thermo Fischer Scientific, Waltham, MA, USA), supplemented with: serum, depleted serum, and purified IgG from ACPA-positive RA patients (n = 3), ACPA-negative RA patients (n = 3), and healthy individuals (n = 3) to a final concentration of 100 μg/ml.
NETs were quantified after IHC staining with mouse anti-human MPO antibody (Abcam) and rabbit anti-human citH3 antibody (Abcam), or the respective isotype controls, followed by incubation with goat anti-mouse IgG AF555 and goat anti-rabbit IgG AF488 (Invitrogen Life Technologies). DNA was counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich). NETs were visualized by using an Olympus IX81 motorized epifluorescence microscope (Olympus America Inc., Center Valley, PA, USA) in conjunction with an Olympus XM10 monochromatic CCD camera (Olympus) and analyzed with the Olympus CellSens Dimension software (Olympus). A minimum of 20 fields at 10× magnification (at least 500 to 1,000 PMNs) per case was evaluated for MPO/citH3 and DNA co-staining through ImageJ analysis software (National Institutes of Health Image Processing, Bethesda, MD, USA); nuclear phenotypes and NETs were determined, counted, and expressed as percentage of the total area of cells in the fields
[
24].
Protein isolation and Western blot analysis
Total protein was isolated with NucleoSpin TriPrep kit (Macherey-Nagel) from 3 × 10
6 PMNs. Proteins from the nuclear and cytoplasmic fractions were isolated by using the Nuclear and Cytoplasmic Protein Extraction Kit (Thermo Scientific). Western blotting was performed by using AnykD Mini-PROTEAN TGX Gels (Biorad, Hercules, CA, USA) and nylon/nitrocellulose membranes (Biorad). Primary and secondary antibodies used were: rabbit anti-PAD4 (Abcam), rabbit anti-MPO (Cell Signalling Technologies, Beverly, MA, USA), mouse anti-β-Actin (Sigma-Aldrich), goat anti-Mouse and/or anti-Rabbit, human anti-HRP (Southern Biotech). HRP activity was detected by using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). Equal loading was verified by using beta-actin or histone H3, when appropriate. Western blots of citrullinated H3 (citH3) protein were performed according to Shechter
et al.
[
25]. Densitometric analysis and protein quantification of the Western blots was performed by using the ImageJ software.
RNA isolation and quantitative real-time PCR
Total RNA was isolated by using RNeasy Mini Kit (Qiagen). TaqMan real-time quantitative RT-PCR was performed by using the Applied Biosystems StepOne Plus cycler (Applied Biosystems) and TaqMan Gene Expression Assay primer/probe sets (Applied Biosystems) for
ELANE (HS00236952_m1),
MPO (HS00924296_m1),
PADI4 (HS00202612_m1), and β
2-microglobulin
B2M (HS99999907_m1). Data were normalized by using the housekeeping gene
B2M, after a selection procedure involving six different endogenous reference genes, as suggested in the MIQE guidelines
[
26]. Relative values were calculated with 2
-DDCt analysis
[
27].
Statistical analysis
All data are presented as mean ± SEM. Descriptive statistics for continuous parameters consisted of median and range, and categoric variables were expressed as percentages. Comparisons between patients and healthy controls were by the Mann–Whitney U test with a Welch posttest correction. Statistical significance in multiple comparisons was by one-way analysis of variance (ANOVA) with a Dunn posttest correction. P < 0.05 was considered statistically significant.
Receiver operating characteristic (ROC) curves were calculated, and the area under the curve (AUC) with corresponding standard errors of means was calculated. Data were processed in GraphPad Prism version 5.0b for MacOSX (GraphPad Software Inc., San Diego, CA, USA). Analysis of covariance (ANCOVA) was conducted with SPSS version 21.0 statistical software (IBM SPSS Inc., Chicago, IL, USA). Additional professional statistical assistance was provided by A. Schoetzau, Basel.
Discussion
Although PMNs figure prominently in the joint effusions and inflamed synovial tissue of RA patients
[
31], the potential roles of NETotic events in the pathophysiology of this disorder have only recently become a focus of attention
[
13,
14]. These studies indicated that RA-derived PMNs were more prone to undergo NETosis, and that NETs themselves could contribute to the generation of auto-antigens (ACPAs) or be the target of auto-antibodies
[
13,
14].
Our studies, performed independently at a time similar time to these, corroborate that NETosis is enhanced in RA, confirming a possible fundamental role of this phenomenon in the underlying etiology of RA. In addition, we extended these observations by examining for changes in the underlying signal-transduction cascade required for the induction of NETosis. The results show that the propensity of circulatory PMNs in RA patients to undergo NETosis is associated with elevations in members of this cascade, including increased intracellular ROS production, enhanced expression of NE and MPO, increased nuclear translocation of PAD4, and citrullination of histones, notably H3. Consequently, these and other key NETotic pathway elements
[
6] could serve as potential therapeutic targets for interventional strategies.
Furthermore, by examining kinetic changes during extended in vitro culture, different nuclear morphometric characteristics were observed in PMNs from RA cases, with a lower proportion of the classic lobulated phenotype, coupled with a much higher proportion of delobulated cells at the initial time point. Unlike in controls, in which an increase in this population was noted over time, this latter population decreased during in vitro culture in RA PMNs. RA PMNs also progressed more rapidly and extensively to a NETotic-spread phenotype than controls, a finding confirmed by analysis of culture supernatants for the products of NETosis.
Akin to that observed in an array of other pathologic conditions ranging from preeclampsia and SLE to cancer and RA
[
8,
9,
12,
13,
17], PMNs from RA patients exhibited an increased response to further stimulation (for instance, by treatment with IL-8, the phorbol ester PMA, or with LPS). This response is in part mediated via the action of PAD4, as the effect of PMA could be significantly reduced by treatment with Cl-amidine, an inhibitor of PAD4
[
30]. In addition, PMA treatment led to an increased nuclear localization of this enzyme, where it presumably could catalyze a more-extensive citrullination of histone proteins, thereby speeding the induction of NETosis.
Although our data are preliminary, they do suggest that PAD4 is extruded onto the NETs during NETosis, as detected with ELISA technology and, to a lesser extent, by fluorescence microscopy. Such an occurrence would have important implications for the development of anti-PAD4 autoantibodies observed in cases with RA
[
32]. Because the presence of such antibodies precedes the development of RA, our data provide further support that NETs may contribute to the underlying etiology of RA, and may be a relatively early event. As the presence of such anti-PAD4 antibodies has been shown to enhance the enzymatic activity of PAD4 in an extracellular environment by reducing the calcium requirement
[
33], their combination with NETs-associated PAD4 could lead to prodigious quantities of citrullinated autoantigens. In addition, the extracellular presence of PAD4 on NETs may further promote the prodigious generation of citrullinated antigens, because molecular structures involving the attachment of enzymes to DNA lattices have been shown to increase their catalytic activity enormously, and thereby form the basis of nano-machines or nano-factories, generating such autoantigens
[
34].
Although these findings must be verified, and it remains to be ascertained whether extracellular NETs-associated PAD4 is active, these data do support and extend recent reports indicating that NETs can be a source for citrullinated autoantigens, and that they react with ACPA or anti-PAD4 antibodies
[
13,
14,
35]. Taken together, these data provide further evidence concerning a key role for PAD4 in the underlying etiology of RA, and offer a potential explanation for the efficacy of PAD4 inhibitor chloramidine in reducing disease symptoms in collagen-induced rat and murine arthritis models for RA
[
36].
It recently was reported that ACPA or IgM RF led to potent increases in NET formation compared with control IgG
[
13]. In our IgG-depletion experiments on ACPA-positive and -negative RA cases, we observed a marked reduction of NET induction to control levels in both cases, whereas reconstitution of serum with IgG gained from depletion almost completely restored NET induction in the ACPA-positive cases. However, in the ACPA-negative cases, no significant increase followed reconstitution. These results support the notion that ACPAs are important inducers of NET formation in ACPA-positive RA cases, and indicate that other mechanisms, such as IgG complexes similar to those involved in NET induction in SLE
[
8], are operative in ACPA-negative RA. Both mechanisms could lead to a common distal mechanism of induction of arthritis.
The observation that the coagulation of blood samples from RA patients during serum preparation triggers extensive NETosis, evident by increased concentrations of cell-free DNA, nucleosomes, or nucleosome/MPO complexes, may have unexpected clinical ramifications. With a sensitivity of 91% and a specificity of 92%, it is possible that the assessment of serum cell-free nucleosomes may serve to distinguish suspected RA patients from healthy controls with a high degree of specificity. It is of interest that this aspect was not significantly influenced by the ACPA status of the RA patients. As such, this assay may be a useful complementary test to perform in conjunction with current ACPA or RF assays, not only to extend diagnostic accuracy, but also to assist in detecting RA in cases that are either ACPA or RF negative. Similar NET induction by ACPA-positive and -negative RA sera and its abrogation by IgG depletion, as discussed earlier, supports the functional aspect of the nucleosome measurement in RA serum.
In a preliminary series of SLE sera, a small and not statistically significant increase of cell-free nucleosomes over normal controls was observed, indicating a slightly elevated propensity for PMNs from SLE patients to undergo NETosis. This was, however, nowhere near the range seen in RA cases, and failed to reach an ROC AUC considered to be clinically relevant. These aspects must be validated in larger multicenter studies.
Acknowledgements
We are grateful to Peter Erb, Ed Palmer and Alan Tyndall for helpful suggestions and comments. We thank Swarna Machineni for performing the ROS analyses, and Maria Stoikou for assistance with the IgG depletion studies. A. Schoetzau, Eudox, Basel, Switzerland provided statistical supervision. Norman Bandelow, Christoph Hemmeler, Eric Deman, Rheumaklinik, Kantonsspital Aarau, and Robyn Benz, Department of Rheumatology, University Hospital, Basel, provided patient samples.
This project was supported by a grant from the Fonds W & W of the Cantonal Hospital Aarau. The position of Chanchal Sur Chowdhury was supported by the University Women's Hospital Basel.
Competing interests
A patent filing has been submitted by the University of Basel and the Cantonal Hospital Aarau for tests developed during this research. We have no other interests to declare.
Authors’ contributions
CSC and SG carried out molecular, cellular studies, immunoassays, and assisted with the manuscript draft. SG conducted in vitro depletion experiments, performed immune histochemical analyses, morphometric analyses, statistical analysis and contributed to writing the manuscript. UW and AB participated in the design of the study, assisted with stratification of patients and healthy donor controls, and assisted with the manuscript draft. SH and PH conceived the study, participated in its design, coordination and wrote the manuscript. All authors read and approved the final manuscript.