Introduction
Systemic lupus erythematosus (SLE) is a chronic systemic autoimmune disease affecting several organ systems such as skin, joints, kidneys and central nervous system. Many of the disease manifestations in SLE are related to immune complexes, consisting of autoantibodies and remnants of apoptotic cells [
1]. Apoptotic cells are thought to be a major source of auto-antigens in SLE, partly because of impaired clearance [
2,
3]. Another potential antigen source is the neutrophil extracellular traps (NETs) that consist of chromatin and antimicrobial enzymes released from neutrophils to trap and kill pathogens. Serum from some SLE patients have a reduced ability to degrade NETs [
4,
5].
Polymorphonuclear leukocytes (PMN), such as neutrophils, are produced in the bone marrow and released to circulation. During acute inflammation an increased mobilization of neutrophils from the bone marrow occurs, which can be observed as increased percentage of CD10
−CD16
low neutrophils in peripheral blood [
6,
7]. The role of PMN in chronic inflammation and autoimmunity is coming into focus, and neutrophils have been suggested to be the primary mediators of end organ-damage responding to deposited immune complexes [
8,
9]. PMN are recruited to inflammatory sites, and activated by pro-inflammatory mediators like complement factors, cytokines and chemokines. Upon activation the expression of various surface proteins changes; for example, C5aR and CD62L are down regulated whereas an increase in CD11b expression is observed [
10,
11]. In addition to the changing expression of surface proteins, activated PMN are primed to release granules and produce reactive oxygen species (ROS) by the nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) complex [
12]. ROS are major effector molecules in inflammatory processes and tightly linked to NETs formation. During the last decade, an increasing amount of data support a T-cell regulating role for monocyte and PMN-produced ROS [
13]–[
16]. Furthermore, the association of SLE to polymorphism in
NCF2, encoding a protein in the NADPH oxidase complex, adds support for the importance of ROS in this disease [
17]. Of note, patients with chronic granulomatous disease, lacking a functional NADPH oxidase complex, show autoimmune features such as high levels of immunoglobulins and autoantibodies, as well as an increased risk of Crohn’s disease and discoid lupus [
18,
19].
This study aims at characterizing PMN from SLE patients (SLE-PMN), in regard to function, bone marrow release and activation to gain knowledge of the role of PMN in SLE and autoimmunity.
Methods
Patients and controls
SLE patients (n = 107) were recruited to the study, when coming to their scheduled visit at the Department of Rheumatology, Skåne University Hospital, Lund, Sweden. All patients fulfilled at least four American College of Rheumatology classification criteria for SLE [
20]. Disease activity was assessed using the systemic lupus erythematosus disease activity index 2000 (SLEDAI-2 K) [
21], and organ damage was evaluated according to the Systemic Lupus International Collaborative Clinics/American College of Rheumatology damage index (SLICC/ACR-DI) [
22]. Demographic and clinical characteristics are shown in Table
1. Healthy blood donors (n = 38, Blood centre in Lund) and healthy volunteers (n = 15) were recruited as controls; ages 18 to 65 years. Complement proteins and autoantibodies were measured using routine analyses (Clinical Immunology and Transfusion Medicine, University and Regional laboratories, Region Skåne, Lund Sweden). The study was approved by the Regional Ethics Review Board at Lund University (file number LU 2010-708) and informed consent was obtained from all participants.
Table 1
Patients characteristics and demographics
Age, median (range) years | 48 (22 to 84) | 43 (22 to 79) | 60 (24 to 84) |
Female gender, n (%) | 81 (88%) | 37 (88%) | 44 (88%) |
Disease duration, median (range) years | 14 (0 to 51) | 9 (0 to 29) | 19 (0 to 51) |
SLEDAI, median (range) | 2 (0 to 16) | 2 (0 to 16) | 1 (0 to 13) |
SLICC/ACR-DI median (range) | 1 (0 to 8) | 0 | 2 (1 to 8) |
PMN 109/L median (range) | 4.0 (<0.1 to 11) | 3.6 (1.4 to 9.8) | 4.7 (<0.1 to 11) |
Disease manifestations at time of sampling, n
| | | |
Lupus headache | 1 | 0 | 1 |
Arthritis | 10 | 5 | 5 |
Kidney involvement (urinary cast, hematuria, proteinuria, or pyuria) | 6 | 3 | 3 |
Rash | 4 | 2 | 2 |
Alopecia | 2 | 1 | 1 |
Low complement (C3 or C4) | 36 | 21 | 15 |
Anti-double stranded DNA antibodies | 18 | 10 | 8 |
Leukopenia | 7 | 4 | 3 |
Treatment
| | | |
Prednisone, % (median dose of treated patients) | 53 (5 mg) | 60 (5 mg) | 50 (6.25 mg) |
Hydroxychloroquine, % (n) | 59 (54) | 69 (29) | 50 (25) |
Chloroquine phosphate, % (n) | 2 (2) | 2 (1) | 2 (1) |
Azathioprine, % (n) | 24 (22) | 29 (12) | 20 (10) |
Mycophenolate mofetil%, (n) | 12 (11) | 14 (6) | 10 (5) |
Rituximab, % (n) | 2 (2) | 0 (0) | 4 (2) |
Methotrexates, % (n) | 4 (4) | 5 (2) | 4 (2) |
Cyclosporine A, % (n) | 2 (2) | 0 (0) | 4 (2) |
Oxidative burst and expression of surface markers
ROS production in peripheral blood PMN was investigated using the PhagoBurst assay, Glycotope Biotechnology, GmBH, Germany, according to the manufacturer’s protocol after activation with phorbol 12-myristate 13-acetate (PMA) or opsonised Escherichia coli (E. coli), and analysed using flow cytometry. At least 15,000 PMN were analysed based on forward and side scatter properties. No patient with ROS deficiency was observed.
ROS formation in peripheral blood PMN was also quantified by oxidation of 2,7-dichlorofluorescein-diacetate (DCFH-DA, Sigma-Aldrich®, St. Louis, MO, USA), as previously described [
23]. As stimuli PMA and
E. coli from the PhagoBurst kit or
Staphylococcus aureus (ATCC 25923, 1 leukocyte: 2,000 bacterial cells) and
Pseudomonas aeruginosa (ATCC 27853, 1 leukocyte: 200 bacterial cells) were used.
S. aureus and
P. aeruginosa were grown in liquid Tryptic Soy Broth (TSB) medium overnight at 37°C and killed by heat (60°C) for 2 h. To confirm bacterial inactivation a sample was inoculated in TSB and kept for 48 h. The bacteria were centrifuged and re-suspended in 0.8% saline. Optical density was adjusted to 24 × 10
8 colony forming units/mL by comparing turbidity to a McFarland scale number 8 BaSO
4 standard solution. DCFH-DA was added to heparinised whole blood before the various stimuli, and then the samples were incubated in a 37°C water bath for 30 minutes. Cells were analysed using flow cytometry.
The expression of selected surface markers on PMN was analysed using flow cytometry. Briefly, peripheral blood was lysed using 0.84% ammonium chloride. The remaining leukocytes were stained for surface expression of CD14 (to exclude monocytes), CD10, CD11b, CD16, CD62L, and C5aR (CD88) (BD Bioscience San Jose, CA, USA). For flow cytometry analysis a FACSCanto II and the DIVA software (Becton Dickinson, BD, New York, NY, USA) were used.
Cell separation and phagocytosis of antibody-coated necrotic cell material by PMN
PMN and peripheral blood mononuclear cells were isolated from heparinised blood of SLE patients by density gradient centrifugation on Polymorphprep™ (Axis-Shield Poc AS, Norway). To obtain necrotic cell material, mononuclear cells were incubated for 10 minutes at 70°C and stained with propidium iodide (BD Bioscience). The propidium iodide-labelled necrotic cell material (4.5 × 105 cells) was then incubated with or without an anti-nucleosome antibody (clone PL2-3; gift from Marc Monestier, Temple University, Philadelphia, USA) at room temperature for 20 minutes. Normal human serum was used as the negative control. The autologous PMN were stained with anti-CD45-FITC (BD Bioscience), and then added to the necrotic cell material, at a concentration of 1.0 × 106 cells/mL in a total volume of 300 μL, followed by incubation at 37°C for 15 minutes. Cells were washed with phosphate-buffered saline pH 7.2 containing 0.1% human serum albumin (Sigma-Aldrich, St. Louis, MO, USA) before analysis by flow cytometry.
Statistical analysis
Correlations were determined by Spearman’s correlation test. The Mann-Whitney U-test was used for two-group comparisons and Kruskal-Wallis test with Dunn’s multiple comparison test was used for three-group comparisons. All P-values were considered significant at P <0.05.
Discussion
PMN were characterized with respect to function, bone marrow release and activation to study their role in SLE, yielding evidence for decreased ROS production in SLE and autoimmunity. Our data support that SLE-PMN have decreased capacity to produce ROS
ex vivo. The association with disease severity, defined as organ damage, further strengthened our finding. Low ROS production has been associated with disease severity of other autoimmune conditions, including Behcet’s disease [
24], Guillain-Barre syndrome [
25] and multiple sclerosis [
26], and might be a common denominator important in the pathogenesis of autoimmunity.
Interestingly, PMA-induced ROS production was significantly reduced in patients with severe disease. However, neither ROS production after
E. coli activation nor phagocytosis of necrotic cell material were associated with organ damage, suggesting that decreased ROS levels after PMA activation is not a sign of impaired PMN functions in general but rather a sign for changed PMN behaviour. While the activation and control of the NADPH oxidase in neutrophils (NOX2) is incompletely understood, it seems that different agonists encountered by the neutrophils engage various combinations of kinases and thereby affect the degree of activity of the NADPH complex, and in the end the amount of ROS produced [
27]. To some extent, this could explain why ROS production after
E. coli activation was not associated with organ damage;
E. coli induced a lower degree of phosphorylation of the NADPH complex regulating subunits compared with PMA that is known to push the NADPH complex to its maximal capacity [
27]. Hence, PMA revealed altered behaviour in PMN from patients with organ damage.
While no association between ROS levels and current disease activity was observed, most patients were in remission or had low to moderate activity based on SLEDAI-2 K (Table
1). An association between disease activity and ROS production could not be excluded based on the available data. The literature is not concurrent regarding ROS production by SLE-PMN [
23,
28,
29]. For example, Perazzio
et al. have shown that neutrophils from SLE patients have an increased capacity to produce ROS, and they did not find any correlation with organ damage or disease activity [
23]. This discrepancy does not reflect the use of different methods, as we observed comparable results with both methods. A more likely explanation is variations in patient cohorts. We have observed an association between decreased ROS formation and disease severity, and a tendency towards increased ROS formation in SLE-PMN in patients with clinical symptoms. Most patients in our study were in remission and possibly our cohort contained more patients with organ damage giving rise to the divergent results. In addition, an influence of genetic factors could not be excluded.
Corticosteroids have been reported to affect the ROS production in PMN in a cumulative dose-dependent way [
30], and it is presently unclear whether this effect is due to increased disease severity. In our study, no correlation between corticosteroid dose and the amount of intracellular ROS produced was observed. The patients had relatively low doses of corticosteroids (mean = 5 mg oral prednisone per day in treated patients) that are likely too low to affect the function of PMN. This could explain why no correlation with ROS levels was found. Moreover, other forms of immune suppressive drugs did neither seem to affect ROS production in the current setting.
Decreased neutrophil counts occur in SLE [
31,
32]. While this is partly due to autoantibodies, there is also evidence for direct effects on the bone marrow production of PMN. Bone marrow from SLE patients has decreased granulocyte-macrophage colony-forming units [
31]–[
33], and we show here that SLE patients have reduced numbers of newly released CD10
−CD16
low neutrophils [
6,
7]. In agreement with earlier observations, these findings suggest an SLE-associated effect on the bone marrow with decreased release of new incompletely differentiated neutrophils. Hence, a decreased number of PMN will be found in the circulation, and with decreased numbers of PMN in the circulation, a prolonged half-life of the existing cells likely occur.
Another possibility is that the PMN phenotype in SLE patients is altered via an as-yet unidentified mechanism. The CD10 and CD16 molecules are normally stored intracellularly and can be rapidly mobilized to the cell surface upon activation [
34]. Hence, an increased percentage of CD10
+CD16
+ cells and a corresponding decrease in CD10
−CD16
low cells could reflect increased activation of PMN
in vivo in SLE. In addition, the percentage of C5aR was decreased, indicating that PMN are activated in peripheral blood [
35,
36]. However, no increase in CD11b expression and corresponding decrease in CD62L were observed on SLE-PMN. Taken together, the observed altered PMN phenotype could be due to prolonged turnover of SLE-PMN in the circulation that gives rise to functional changes such as decreased ROS production and an atypical expression of surface markers.
Acknowledgements
This work was supported by grants from Alfred Österlund’s Foundation, The Crafoord Foundation, Greta and Johan Kock’s Foundation, King Gustaf V’s 80th Birthday Foundation, Lund University Hospital, the Swedish Rheumatism Association, the Swedish Research council (X65X-15152) and the Foundation of the National Board of Health and Welfare.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
ÅP, BG and ÅJ did the laboratory work. BG and AB gathered all clinical data. AB, TH, MH, SW, BG and ÅJ contributed to the design of the study and wrote the manuscript. All authors read and approved the final manuscript.