Low production of reactive oxygen species in granulocytes is associated with organ damage in systemic lupus erythematosus
© Bengtsson et al.; licensee BioMed Central Ltd. 2014
Received: 3 December 2013
Accepted: 29 May 2014
Published: 5 June 2014
Polymorphonuclear leukocytes (PMN) are main effector cells in the acute immune response. While the specific role of PMN in systemic lupus erythematosus (SLE) and autoimmunity is still unclear, their importance in chronic inflammation is gaining more attention. Here we investigate aspects of function, bone marrow release and activation of PMN in patients with SLE.
The following PMN functions and subsets were evaluated using flow cytometry; (a) production of reactive oxygen species (ROS) after ex vivo stimulation with phorbol 12-myristate 13-acetate (PMA) or Escherichia coli (E. coli); (b) capacity to phagocytose antibody-coated necrotic cell material; (c) PMN recently released from bone marrow, defined as percentage of CD10−D16low in peripheral blood, and (d) PMN activation markers; CD11b, CD62L and C5aR.
SLE patients (n = 92) showed lower ROS production compared with healthy controls (n = 38) after activation ex vivo. The ROS production was not associated with corticosteroid dose or other immunotherapies. PMA induced ROS production was significantly reduced in patients with severe disease. In contrast, neither ROS levels after E. coli activation, nor the capacity to phagocytose were associated with disease severity. This suggests that decreased ROS production after PMA activation is a sign of changed PMN behaviour rather than generally impaired functions. The CD10−CD16low phenotype constitute 2% of PMN in peripheral blood of SLE patients compared with 6.4% in controls, indicating a decreased release of PMN from the bone marrow in SLE. A decreased expression of C5aR on PMN was observed in SLE patients, pointing towards in vivo activation.
Our results indicate that PMN from SLE patients have altered function, are partly activated and are released abnormally from bone marrow. The association between low ROS formation in PMN and disease severity is consistent with findings in other autoimmune diseases and might be considered as a risk factor.
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 . 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−CD16low 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 . 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 –. 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 . 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.
Patients and controls
Patients characteristics and demographics
All patients (n = 92)
No organ damage SLICC/ACR-DI = 0 (n = 42)
Organ damage SLICC/ACR-DI ≥1 (n = 50)
Age, median (range) years
48 (22 to 84)
43 (22 to 79)
60 (24 to 84)
Female gender, n (%)
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)
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
Kidney involvement (urinary cast, hematuria, proteinuria, or pyuria)
Low complement (C3 or C4)
Anti-double stranded DNA antibodies
Prednisone, % (median dose of treated patients)
53 (5 mg)
60 (5 mg)
50 (6.25 mg)
Hydroxychloroquine, % (n)
Chloroquine phosphate, % (n)
Azathioprine, % (n)
Mycophenolate mofetil%, (n)
Rituximab, % (n)
Methotrexates, % (n)
Cyclosporine A, % (n)
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 . 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 × 108 colony forming units/mL by comparing turbidity to a McFarland scale number 8 BaSO4 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.
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.
Decreased production of ROS in SLE-PMN
Comparisons between the PhagoBurst and the DCFH-DA assay
Relative reactive oxygen species formation in SLE patients as% of formation in healthy controls
68 ± 7.7
76 ± 6.2
84 ± 13
103 ± 13
92 ± 8.7
102 ± 6.3
88 ± 12
97 ± 14
Organ damage was associated with low ROS production in SLE-PMN
Next, we investigated if disease activity, at the time point of sampling, was associated with ROS production. The patients were divided into three groups based on the SLEDAI-2 K : (1) no activity, (2) laboratory parameters only, such as low complement and anti-double stranded DNA antibodies; and (3) clinical manifestations, for example, nephritis, rash and arthritis. No association between ROS production and disease activity (P = 0.0654) was observed (Figure 3B).
Phagocytosis of antibody-coated microbes and foreign material precedes ROS production in PMN. To evaluate further the function of PMN in SLE, in particular in patients with organ damage, the phagocytosis capacity was investigated in 40 out of the 92 patients. Antibody-coated necrotic cells were chosen as stimuli for phagocytosis to relate to lupus erythematosus cells, for example, PMN containing phagocytosed antibody-coated dead cell materials, a phenomenon almost pathognomonic for SLE. No differences were observed between patients with (n = 25; SLICC/ACR-DI ≥1), compared to patients without organ damage (n = 15; SLICC/ACR-DI = 0), further suggesting that the decreased ROS production in patients with severe disease is not due to a general unresponsiveness (Figure 3C). No associations between disease activity based on SLEDAI-2 K and the ability to phagocytose were observed (not shown).
Low numbers of CD10−CD16low SLE-PMN
Decreased expression of C5aR (CD88) on polymorphonuclear leukocytes (PMN) from patients with systemic lupus erythematosus (SLE)
Healthy blood donors
92 ± 3.8
77 ± 1
466 ± 24
285 ± 4
98 ± 0.3
98 ± 0.2
1180 ± 64
1100 ± 42
100 ± 0
100 ± 0
9343 ± 578
8166 ± 270
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 , Guillain-Barre syndrome  and multiple sclerosis , 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 . 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 . 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 . 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 , 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 –, and we show here that SLE patients have reduced numbers of newly released CD10−CD16low 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 . Hence, an increased percentage of CD10+CD16+ cells and a corresponding decrease in CD10−CD16low 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.
Our study shows an association between low ROS formation and disease severity in SLE. This is consistent with findings in other autoimmune disease, suggesting that a decrease in NADPH complex-mediated ROS production is a risk factor in autoimmunity. The phenotype observed in SLE-PMN could be due to aberrant production of leukocytes in the bone marrow and/or in vivo activation in the circulation. Future studies will illuminate the role of ROS formation and PMN in SLE and autoimmunity.
- E. coli:
nicotinamide adenine dinucleotide phosphate-oxidase
Neutrophil extracellular traps
Phorbol 12-myristate 13-acetate
reactive oxygen species
systemic lupus erythematosus
- SLEDAI-2 K:
systemic lupus erythematosus disease activity index 2000
PMN from SLE patients
Systemic Lupus International Collaborative Clinics/American College of Rheumatology damage index.
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.
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