Low production of reactive oxygen species in granulocytes is associated with organ damage in systemic lupus erythematosus
Bengtsson et al. Arthritis Research & Therapy 2014, 16:R120http://arthritis-research.com/content/16/3/R120
Low production of reactive oxygen species ingranulocytes is associated with organ damage insystemic lupus erythematosus
Anders A Bengtsson1, Åsa Pettersson2, Stina Wichert3, Birgitta Gullstrand4, Markus Hansson3,Thomas Hellmark2 and Åsa CM Johansson3,5*
Introduction: Polymorphonuclear leukocytes (PMN) are main effector cells in the acute immune response. Whilethe specific role of PMN in systemic lupus erythematosus (SLE) and autoimmunity is still unclear, their importancein chronic inflammation is gaining more attention. Here we investigate aspects of function, bone marrow releaseand activation of PMN in patients with SLE.
Methods: The following PMN functions and subsets were evaluated using flow cytometry; (a) production ofreactive 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 bonemarrow, defined as percentage of CD10−D16low in peripheral blood, and (d) PMN activation markers; CD11b, CD62Land C5aR.
Results: SLE patients (n = 92) showed lower ROS production compared with healthy controls (n = 38) afteractivation ex vivo. The ROS production was not associated with corticosteroid dose or other immunotherapies. PMAinduced ROS production was significantly reduced in patients with severe disease. In contrast, neither ROS levelsafter E. coli activation, nor the capacity to phagocytose were associated with disease severity. This suggests thatdecreased ROS production after PMA activation is a sign of changed PMN behaviour rather than generally impairedfunctions. The CD10−CD16low phenotype constitute 2% of PMN in peripheral blood of SLE patients compared with6.4% in controls, indicating a decreased release of PMN from the bone marrow in SLE. A decreased expression ofC5aR on PMN was observed in SLE patients, pointing towards in vivo activation.
Conclusions: Our results indicate that PMN from SLE patients have altered function, are partly activated and arereleased abnormally from bone marrow. The association between low ROS formation in PMN and disease severity isconsistent with findings in other autoimmune diseases and might be considered as a risk factor.
major source of auto-antigens in SLE, partly because of
Systemic lupus erythematosus (SLE) is a chronic systemic
impaired clearance . Another potential antigen source
autoimmune disease affecting several organ systems such
is the neutrophil extracellular traps (NETs) that consist of
as skin, joints, kidneys and central nervous system. Many
chromatin and antimicrobial enzymes released from neu-
of the disease manifestations in SLE are related to immune
trophils to trap and kill pathogens. Serum from some SLE
complexes, consisting of autoantibodies and remnants of
patients have a reduced ability to degrade NETs .
apoptotic cells ]. Apoptotic cells are thought to be a
Polymorphonuclear leukocytes (PMN), such as neu-
trophils, are produced in the bone marrow and releasedto circulation. During acute inflammation an increased
* Correspondence: 3Department of Haematology, Lund University, BMC B13, 221 84 Lund,
mobilization of neutrophils from the bone marrow oc-
curs, which can be observed as increased percentage of
5Clinical Immunology and Transfusion Medicine, University and Regional
CD10−CD16low neutrophils in peripheral blood . The
Laboratories Region Skåne, 221 85 Lund, SwedenFull list of author information is available at the end of the article
role of PMN in chronic inflammation and autoimmunity
2014 Bengtsson et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of theCreative Commons Attribution License which permits unrestricted use,distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons PublicDomain Dedication waiver applies to the data made available in thisarticle, unless otherwise stated.
Bengtsson et al. Arthritis Research & Therapy 2014, 16:R120
is coming into focus, and neutrophils have been suggested
patients with chronic granulomatous disease, lacking a
to be the primary mediators of end organ-damage res-
functional NADPH oxidase complex, show autoimmune
ponding to deposited immune complexes PMN are
features such as high levels of immunoglobulins and auto-
recruited to inflammatory sites, and activated by pro-
antibodies, as well as an increased risk of Crohn's disease
inflammatory mediators like complement factors, cytokines
and discoid lupus .
and chemokines. Upon activation the expression of various
This study aims at characterizing PMN from SLE pa-
surface proteins changes; for example, C5aR and CD62L
tients (SLE-PMN), in regard to function, bone marrow
are down regulated whereas an increase in CD11b expres-
release and activation to gain knowledge of the role of
sion is observed [In addition to the changing expres-
PMN in SLE and autoimmunity.
sion of surface proteins, activated PMN are primed torelease granules and produce reactive oxygen species (ROS)
by the nicotinamide adenine dinucleotide phosphate-
Patients and controls
oxidase (NADPH) complex . ROS are major effector
SLE patients (n = 107) were recruited to the study, when
molecules in inflammatory processes and tightly linked to
coming to their scheduled visit at the Department of
NETs formation. During the last decade, an increasing
Rheumatology, Skåne University Hospital, Lund, Sweden.
amount of data support a T-cell regulating role for mono-
All patients fulfilled at least four American College of
cyte and PMN-produced ROS [–]. Furthermore, the
Rheumatology classification criteria for SLE Disease
association of SLE to polymorphism in NCF2, encoding a
activity was assessed using the systemic lupus erythemato-
protein in the NADPH oxidase complex, adds support for
sus disease activity index 2000 (SLEDAI-2 K) , and
the importance of ROS in this disease Of note,
organ damage was evaluated according to the Systemic
Table 1 Patients characteristics and demographics
SLICC/ACR-DI = 0 (n = 42)
SLICC/ACR-DI ≥1 (n = 50)
Age, median (range) years
Female gender, n (%)
Disease duration, median (range) years
SLEDAI, median (range)
SLICC/ACR-DI median (range)
PMN 109/L median (range)
4.0 (<0.1 to 11)
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)
Hydroxychloroquine, % (n)
Chloroquine phosphate, % (n)
Azathioprine, % (n)
Mycophenolate mofetil%, (n)
Methotrexates, % (n)
Cyclosporine A, % (n)
SLICC/ACR-DI, Systemic Lupus International Collaborative Clinics/American College of Rheumatology (ACR) damage index; SLEDAI-2 K, Systemic lupus erythematosusdisease activity index 2000; PMN, polymorphonuclear leukocytes.
Bengtsson et al. Arthritis Research & Therapy 2014, 16:R120
Lupus International Collaborative Clinics/American Col-
Poc AS, Norway). To obtain necrotic cell material, mono-
lege of Rheumatology damage index (SLICC/ACR-DI) ].
nuclear cells were incubated for 10 minutes at 70°C and
Demographic and clinical characteristics are shown in
stained with propidium iodide (BD Bioscience). The propi-
Table Healthy blood donors (n = 38, Blood centre in
dium iodide-labelled necrotic cell material (4.5 × 105 cells)
Lund) and healthy volunteers (n = 15) were recruited as
was then incubated with or without an anti-nucleosome
controls; ages 18 to 65 years. Complement proteins and
antibody (clone PL2-3; gift from Marc Monestier, Temple
autoantibodies were measured using routine analyses
University, Philadelphia, USA) at room temperature for
(Clinical Immunology and Transfusion Medicine, University
20 minutes. Normal human serum was used as the nega-
and Regional laboratories, Region Skåne, Lund Sweden).
tive control. The autologous PMN were stained with anti-
The study was approved by the Regional Ethics Review
CD45-FITC (BD Bioscience), and then added to the nec-
Board at Lund University (file number LU 2010-708) and
rotic cell material, at a concentration of 1.0 × 106 cells/mL
informed consent was obtained from all participants.
in a total volume of 300 μL, followed by incubation at37°C for 15 minutes. Cells were washed with phosphate-
Oxidative burst and expression of surface markers
buffered saline pH 7.2 containing 0.1% human serum
ROS production in peripheral blood PMN was investigated
albumin (Sigma-Aldrich, St. Louis, MO, USA) before ana-
using the PhagoBurst assay, Glycotope Biotechnology,
lysis by flow cytometry.
GmBH, Germany, according to the manufacturer's proto-col after activation with phorbol 12-myristate 13-acetate
(PMA) or opsonised Escherichia coli (E. coli), and analysed
Correlations were determined by Spearman's correlation
using flow cytometry. At least 15,000 PMN were analysed
test. The Mann-Whitney U-test was used for two-group
based on forward and side scatter properties. No patient
comparisons and Kruskal-Wallis test with Dunn's mul-
with ROS deficiency was observed.
tiple comparison test was used for three-group compari-
ROS formation in peripheral blood PMN was also quan-
sons. All P-values were considered significant at P <0.05.
tified by oxidation of 2,7-dichlorofluorescein-diacetate(DCFH-DA, Sigma-Aldrich®, St. Louis, MO, USA), as previ-
ously described . As stimuli PMA and E. coli from the
Decreased production of ROS in SLE-PMN
PhagoBurst kit or Staphylococcus aureus (ATCC 25923, 1
Phagocyte-produced ROS are important effector mole-
leukocyte: 2,000 bacterial cells) and Pseudomonas aerugi-
cules in the defence against microbes, and could also be
nosa (ATCC 27853, 1 leukocyte: 200 bacterial cells) were
involved in the regulation of the adaptive immune system
used. S. aureus and P. aeruginosa were grown in liquid
. To evaluate PMN function in SLE, we decided to in-
Tryptic Soy Broth (TSB) medium overnight at 37°C and
vestigate intracellular ROS production. PMN in peripheral
killed by heat (60°C) for 2 h. To confirm bacterial inactiva-
whole blood from SLE patients (n = 92, Table and
tion a sample was inoculated in TSB and kept for 48 h.
healthy controls (n = 38), were stimulated with either the
The bacteria were centrifuged and re-suspended in 0.8%
protein kinase C activator, PMA, or with opsonised E. coli.
saline. Optical density was adjusted to 24 × 108 colony form-
SLE-PMN showed a decreased capacity to produce ROS
ing units/mL by comparing turbidity to a McFarland scale
ex vivo after activation with both PMA (P <0.0001) and E.
number 8 BaSO4 standard solution. DCFH-DA was added
coli (P = 0.0002) (Figure The decreased amount of ROS
to heparinised whole blood before the various stimuli, and
produced by SLE-PMN was not associated with the dose
then the samples were incubated in a 37°C water bath for
of prednisone or hydroxychloroquine treatment (Figure
30 minutes. Cells were analysed using flow cytometry.
and B) or other immune suppressive drugs listed in Table
The expression of selected surface markers on PMN was
(not shown). Perazzio et al. have previously shown an in-
analysed using flow cytometry. Briefly, peripheral blood
creased ROS production in SLE-PMN after in vitro activa-
was lysed using 0.84% ammonium chloride. The remaining
tion with S. aureus or P. aeruginosa using DCFH-DA as
leukocytes were stained for surface expression of CD14 (to
fluorochrome . To evaluate whether this discrepancy
exclude monocytes), CD10, CD11b, CD16, CD62L, and
was due to experimental procedure or differences in pa-
C5aR (CD88) (BD Bioscience San Jose, CA, USA). For flow
tient population, patients (n = 15) and controls (n = 15)
cytometry analysis a FACSCanto II and the DIVA software
were analysed in parallel with both methods, using S.
(Becton Dickinson, BD, New York, NY, USA) were used.
aureus, P. aeruginosa, PMA and E. coli as stimuli. Similarfindings where observed between the two methods
Cell separation and phagocytosis of antibody-coated
(Table . SLE-PMN showed a decreased intracellular
necrotic cell material by PMN
ROS formation after PMA activation compared with con-
PMN and peripheral blood mononuclear cells were iso-
trols (PhagoBurst test: P = 0.0394 and DCFH-DA: P =
lated from heparinised blood of SLE patients by density
0.0146) whereas no significant difference was observed
gradient centrifugation on Polymorphprep™ (Axis-Shield
with the other stimuli (not shown). The decreased ROS
Bengtsson et al. Arthritis Research & Therapy 2014, 16:R120
Figure 1 Polymorphonuclear leukocytes (PMN) from patients with
systemic lupus erythematosus (SLE) produced fewer reactive
oxygen species (ROS) than PMN from healthy blood donors. Thecapacities of PMN from healthy blood donors (HBD), n = 38, and SLE
patients, n = 92, to produce ROS upon activation with phorbol
12-myristate 13-acetate or opsonised E. coli were investigated using flowcytometry. The amount of ROS produced is shown as geometric meanfluorescence intensity (geo mean fluorescence, MFI). The two-sided
Mann-Whitney test was used to calculate the level of significance. Thehorizontal lines represent the median value of each dataset.
production in our examined SLE cohort after PMA activa-
tion was consistent using both methods in contrast to thefindings of Perazzio et al., suggesting differences in patient
Prednisone dose pe rday
Organ damage was associated with low ROS production
Figure 2 Reactive oxygen species (ROS) production in
polymorphonuclear leukocytes (PMN) from systemic lupus
The severity of autoimmune diseases has previously been
erythematosus (SLE) patients did not correlate with
associated with decreased ROS production –. Hence,
Hydroxychloroquine treatment or the dose of prednisone. ROS
to study if the severity of SLE was associated with changes
production was measured by flow cytometry after ex vivo activationof PMN with phorbol 12-myristate 13-acetate (A) the amount ROS
in ROS production, the patients were divided in two
produced by SLE-PMN, treated with or without Hydroxychloroquine.
groups based on the presence of organ damage or not ac-
(B) The amount of ROS produced in individual patients plotted
cording to SLICC/ACR-DI (Table . PMN from patients
against the dose of prednisone at the day of sampling. The amount
with SLICC/ACR-DI ≥1 had decreased ROS production,
ROS produced is shown as geometric mean fluorescence intensity
compared with patients without organ damage (Figure ),
(geo mean fluorescence, MFI). The line represents the median valueof each dataset.
when activated with PMA (P = 0.0022). No difference wasseen after activation with E. coli (not shown). Patients withorgan damage were in general older than patients without
ROS production and disease activity (P = 0.0654) was ob-
(Table however, the age of the patients was not cor-
served (Figure B).
related with PMA-induced oxidative burst in PMN (not
Phagocytosis of antibody-coated microbes and foreign
material precedes ROS production in PMN. To evaluate
Next, we investigated if disease activity, at the time point
further the function of PMN in SLE, in particular in pa-
of sampling, was associated with ROS production. The
tients with organ damage, the phagocytosis capacity was
patients were divided into three groups based on the
investigated in 40 out of the 92 patients. Antibody-coated
SLEDAI-2 K [(1) no activity, (2) laboratory parameters
necrotic cells were chosen as stimuli for phagocytosis to
only, such as low complement and anti-double stranded
relate to lupus erythematosus cells, for example, PMN
DNA antibodies; and (3) clinical manifestations, for ex-
containing phagocytosed antibody-coated dead cell mate-
ample, nephritis, rash and arthritis. No association between
rials, a phenomenon almost pathognomonic for SLE. No
Bengtsson et al. Arthritis Research & Therapy 2014, 16:R120
Table 2 Comparisons between the PhagoBurst and the DCFH-DA assay
Relative reactive oxygen species formation in SLE patients as% of formation in healthy controls
Comparisons of the PhagoBurst assay with the dichlorodihydrofluorescein-diacetate (DCFH-DA) assay according to Perazzio et al. [No significant differencesbetween the methods were observed. Polymorphonuclear leukocytes (PMN) from systemic lupus erythematosus (SLE) patients (n = 15) and healthy controls(n = 15) were analysed in parallel with both methods using phorbol 12-myristate 13-acetate (PMA), E. coli, S. aureus or P. aeruginosa as stimuli. ROS formation wasdefined as geometric mean fluorescence intensity. Samples from each individual patient were divided by the mean value of the controls to gain the relative ROSformation in SLE-PMN as% of ROS produced in healthy controls. Values represent mean ± standard error of the mean. The two-sided Mann-Whitney test was usedto calculate the level of significance.
differences were observed between patients with (n =
conditions, including Behcet's disease , Guillain-Barre
25; SLICC/ACR-DI ≥1), compared to patients without
syndrome and multiple sclerosis , and might be a
organ damage (n = 15; SLICC/ACR-DI = 0), further sug-
common denominator important in the pathogenesis of
gesting that the decreased ROS production in patients
with severe disease is not due to a general unrespon-
Interestingly, PMA-induced ROS production was signifi-
siveness (Figure ). No associations between disease
cantly reduced in patients with severe disease. However,
activity based on SLEDAI-2 K and the ability to phago-
neither ROS production after E. coli activation nor phago-
cytose were observed (not shown).
cytosis of necrotic cell material were associated with organdamage, suggesting that decreased ROS levels after PMA
Low numbers of CD10−CD16low SLE-PMN
activation is not a sign of impaired PMN functions in gen-
During acute inflammation, an increased percentage of
eral but rather a sign for changed PMN behaviour. While
CD10−CD16low neutrophils in peripheral blood are thought
the activation and control of the NADPH oxidase in neu-
to reflect an increased mobilization of cells from bone
trophils (NOX2) is incompletely understood, it seems that
marrow . To study the frequency of newly released
different agonists encountered by the neutrophils engage
CD10−CD16low PMN in peripheral blood, samples from
various combinations of kinases and thereby affect the de-
73 SLE patients, and 27 healthy controls were analysed by
gree of activity of the NADPH complex, and in the end
flow cytometry. SLE patients showed lower percentages of
the amount of ROS produced . To some extent, this
CD10−CD16low PMN (Figure ), compared with healthy
could explain why ROS production after E. coli activation
controls (P <0.0001). Hence, the SLE-PMN were to a high
was not associated with organ damage; E. coli induced a
extent CD10+CD16+ compared with controls (P <0.0001),
lower degree of phosphorylation of the NADPH complex
which is consistent with a decreased release of PMN from
regulating subunits compared with PMA that is known to
the bone marrow.
push the NADPH complex to its maximal capacity .
To characterize the activation status of PMN in periph-
Hence, PMA revealed altered behaviour in PMN from pa-
eral blood the expression of C5aR, CD11b and CD62L was
tients with organ damage.
investigated. SLE-PMN were to a lesser extent C5aR posi-
While no association between ROS levels and current
tive (P <0.0001), and the positive cells expressed less C5aR
disease activity was observed, most patients were in remis-
(P <0.0001) (Table . No differences in the expression of
sion or had low to moderate activity based on SLEDAI-
CD11b or CD62L were observed (Table , indicating that
2 K (Table . An association between disease activity and
the cells were only partly activated. C5aR expression and
ROS production could not be excluded based on the avail-
percentage of CD10−CD16lowPMN were not correlated
able data. The literature is not concurrent regarding ROS
with SLICC/ACR-DI or SLEDAI-2 K (not shown).
production by SLE-PMN For example, Perazzioet al. have shown that neutrophils from SLE patients have
an increased capacity to produce ROS, and they did not
PMN were characterized with respect to function, bone
find any correlation with organ damage or disease activity
marrow release and activation to study their role in SLE,
. This discrepancy does not reflect the use of different
yielding evidence for decreased ROS production in SLE
methods, as we observed comparable results with both
and autoimmunity. Our data support that SLE-PMN have
methods. A more likely explanation is variations in patient
decreased capacity to produce ROS ex vivo. The associ-
cohorts. We have observed an association between de-
ation with disease severity, defined as organ damage, fur-
creased ROS formation and disease severity, and a ten-
ther strengthened our finding. Low ROS production has
dency towards increased ROS formation in SLE-PMN in
been associated with disease severity of other autoimmune
patients with clinical symptoms. Most patients in our
Bengtsson et al. Arthritis Research & Therapy 2014, 16:R120
Figure 3 Organ damage in systemic lupus erythematosus (SLE)patients is associated with decreased reactive oxygen species
(ROS) production. ROS production was measured by flow cytometry
after ex vivo activation of peripheral blood polymorphonuclearleukocytes (PMN) with phorbol 12-myristate 13-acetate. (A) The amountof ROS produced by PMN from patients with organ damage (Systemic
Lupus International Col aborative Clinics/ACR damage index (SLICC/ACR-DI) ≥1) was compared with PMN from patients without organdamage (SLICC/ACR-DI = 0). (B) The amount of ROS produced by PMNfrom patients with (1) inactive disease (SLE activity index 2000 (SLEDAI-
2 K) = 0) affected laboratory parameters, such as low complement andanti-double stranded DNA antibodies, but no clinical symptoms; (2)
laboratory parameters only, and patients with clinical manifestations,
for example, nephritis, rash and arthritis; and (3) clinical symptoms
(P = 0,0654). (C) Phagocytosis of necrotic cel material, in the presenceof serum and anti-nucleosome antibodies, by purified polymorphnucleated leukocytes (n = 40), was analysed using flow cytometry. The
patients were divided based on organ damage (SLICC/ACR-DI) and
their phagocytosis capacity is shown as% phagocytosing cells. Thetwo-sided Mann-Whitney test was used to calculate the level of
significance between two groups and Kruskal-Wallis test with Dunn'smultiple comparison test was used to calculate the level of significance
between three groups. The line represents the median value of each
dataset. MFI, mean fluorescence intensity.
Lab parameters only
% positive granulocytes
HBD SLE HBD SLE
Figure 4 Decreased numbers of CD10−CD16low
polymorphonuclear leukocytes (PMN) from patients withsystemic lupus erythematosus (SLE). The frequencies of CD10+CD16+(mainly segment nucleated neutrophils) and CD10−CD16low (suggested
as a marker for newly released neutrophils) PMN were investigated in
healthy blood donors (HBD, n = 27) and patients (n = 73) using flowcytometry. The two-sided Mann-Whitney test was used to calculate the
level of significance. The line represents the median value of eachdataset.
Bengtsson et al. Arthritis Research & Therapy 2014, 16:R120
Table 3 Decreased expression of C5aR (CD88) on
was decreased, indicating that PMN are activated in per-
polymorphonuclear leukocytes (PMN) from patients with
ipheral blood ]. However, no increase in CD11b
systemic lupus erythematosus (SLE)
expression and corresponding decrease in CD62L were
Healthy blood donors
observed on SLE-PMN. Taken together, the observed
altered PMN phenotype could be due to prolonged turn-
over of SLE-PMN in the circulation that gives rise to func-
tional changes such as decreased ROS production and an
atypical expression of surface markers.
Our study shows an association between low ROS forma-
Comparisons were done between SLE patients and healthy blood donors in
tion and disease severity in SLE. This is consistent with
regard of the frequency (%) and amount (geometric mean fluorescence intensity
findings in other autoimmune disease, suggesting that a
(geoMFI) of positive cells) of CD88, CD62L and CD11b on CD10+CD16+ PMN.
Values represent mean ± standard error of the mean. The two-sided
decrease in NADPH complex-mediated ROS production
Mann-Whitney test was used to calculate the level of significance; ns, not significant.
is a risk factor in autoimmunity. The phenotype observedin SLE-PMN could be due to aberrant production of leu-
study were in remission and possibly our cohort contained
kocytes in the bone marrow and/or in vivo activation in
more patients with organ damage giving rise to the diver-
the circulation. Future studies will illuminate the role of
gent results. In addition, an influence of genetic factors
ROS formation and PMN in SLE and autoimmunity.
could not be excluded.
Corticosteroids have been reported to affect the ROS
AbbreviationsE. coli: Escherichia coli; NADPH: nicotinamide adenine dinucleotide
production in PMN in a cumulative dose-dependent way
phosphate-oxidase; NETs: Neutrophil extracellular traps; PMA: Phorbol
, and it is presently unclear whether this effect is due
12-myristate 13-acetate; PMN: polymorphonuclear leukocytes; ROS: reactive
to increased disease severity. In our study, no correlation
oxygen species; SLE: systemic lupus erythematosus; SLEDAI-2 K: systemiclupus erythematosus disease activity index 2000; SLE-PMN: PMN from SLE
between corticosteroid dose and the amount of intracellu-
patients; SLICC/ACR-DI: Systemic Lupus International Collaborative Clinics/
lar ROS produced was observed. The patients had rela-
American College of Rheumatology damage index.
tively low doses of corticosteroids (mean = 5 mg oral
prednisone per day in treated patients) that are likely too
The authors declare that they have no competing interests.
low to affect the function of PMN. This could explain whyno correlation with ROS levels was found. Moreover, other
forms of immune suppressive drugs did neither seem to
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
affect ROS production in the current setting.
wrote the manuscript. All authors read and approved the final manuscript.
Decreased neutrophil counts occur in SLE ].
While this is partly due to autoantibodies, there is also evi-
AcknowledgementsThis work was supported by grants from Alfred Österlund's Foundation, The
dence for direct effects on the bone marrow production of
Crafoord Foundation, Greta and Johan Kock's Foundation, King Gustaf V's
PMN. Bone marrow from SLE patients has decreased
80th Birthday Foundation, Lund University Hospital, the Swedish Rheumatism
granulocyte-macrophage colony-forming units ],
Association, the Swedish Research council (X65X-15152) and the Foundationof the National Board of Health and Welfare.
and we show here that SLE patients have reduced num-bers of newly released CD10−CD16low neutrophils [. In
agreement with earlier observations, these findings suggest
Department of Clinical Sciences, Section of Rheumatology, Lund University
and Skåne University Hospital, 221 85 Lund, Sweden. 2Department of
an SLE-associated effect on the bone marrow with de-
Nephrology, Clinical Sciences in Lund, Lund University, BMC B13, 221 84
creased release of new incompletely differentiated neutro-
Lund, Sweden. 3Department of Haematology, Lund University, BMC B13, 221
phils. Hence, a decreased number of PMN will be found
84 Lund, Sweden. 4Department of Laboratory Medicine Lund. Section ofMicrobiology, Immunology and Glycobiology, Lund University, Lund,
in the circulation, and with decreased numbers of PMN in
Sweden. 5Clinical Immunology and Transfusion Medicine, University and
the circulation, a prolonged half-life of the existing cells
Regional Laboratories Region Skåne, 221 85 Lund, Sweden.
Received: 3 December 2013 Accepted: 29 May 2014
Another possibility is that the PMN phenotype in SLE
Published: 5 June 2014
patients is altered via an as-yet unidentified mechanism.
The CD10 and CD16 molecules are normally stored intra-
Bengtsson A, Nezlin R, Shoenfeld Y, Sturfelt G: DNA levels in circulating
cellularly and can be rapidly mobilized to the cell sur-
immune complexes decrease at severe SLE flares-correlation with
face upon activation ]. Hence, an increased percentage
complement component C1q. J Autoimmun 1999, 13:111–119.
of CD10+CD16+ cells and a corresponding decrease in
Herrmann M, Voll RE, Zoller OM, Hagenhofer M, Ponner BB, Kalden JR:Impaired phagocytosis of apoptotic cell material by monocyte-derived
CD10−CD16low cells could reflect increased activation of
macrophages from patients with systemic lupus erythematosus. Arthritis
PMN in vivo in SLE. In addition, the percentage of C5aR
Rheum 1998, 41:1241–1250.
Bengtsson et al. Arthritis Research & Therapy 2014, 16:R120
Gaipl US, Voll RE, Sheriff A, Franz S, Kalden JR, Herrmann M: Impaired
rheumatology damage index for systemic lupus erythematosus. Arthritis
clearance of dying cells in systemic lupus erythematosus. Autoimmun Rev
Rheum 1996, 39:363–369.
Perazzio SF, Salomao R, Silva NP, Andrade LE: Increased neutrophil
Hakkim A, Furnrohr BG, Amann K, Laube B, Abed UA, Brinkmann V,
oxidative burst metabolism in systemic lupus erythematosus. Lupus 2012,
Herrmann M, Voll RE, Zychlinsky A: Impairment of neutrophil extracellular
trap degradation is associated with lupus nephritis. Proc Natl Acad Sci
Eksioglu-Demiralp E, Direskeneli H, Kibaroglu A, Yavuz S, Ergun T, Akoglu T:
USA 2010, 107:9813–9818.
Neutrophil activation in Behcet's disease. Clin Exp Rheumatol 2001,
Leffler J, Martin M, Gullstrand B, Tyden H, Lood C, Truedsson L, Bengtsson
AA, Blom AM: Neutrophil extracellular traps that are not degraded in
Mossberg N, Andersen O, Nilsson S, Dahlgren C, Hellstrand K, Lindh M,
systemic lupus erythematosus activate complement exacerbating the
Svedhem A, Bergstrom T, Movitz C: Oxygen radical production and severity
disease. J Immunol 2012, 188:3522–3531.
of the Guillain–Barre syndrome. J Neuroimmunol 2007, 192:186–191.
Kaneko T, Stearns-Kurosawa DJ, Taylor F Jr, Twigg M, Osaki K, Kinasewitz GT,
Mossberg N, Movitz C, Hellstrand K, Bergstrom T, Nilsson S, Andersen O:
Peer G, Kurosawa S: Reduced neutrophil CD10 expression in nonhuman
Oxygen radical production in leukocytes and disease severity in multiple
primates and humans after in vivo challenge with E. coli or
sclerosis. J Neuroimmunol 2009, 213:131–134.
lipopolysaccharide. Shock 2003, 20:130–137.
El-Benna J, Dang PM, Gougerot-Pocidalo MA: Priming of the neutrophil
Orr Y, Taylor JM, Bannon PG, Geczy C, Kritharides L: Circulating CD10-/
NADPH oxidase activation: role of p47phox phosphorylation and NOX2
CD16low neutrophils provide a quantitative index of active bone
mobilization to the plasma membrane. Semin Immunopathol 2008,
marrow neutrophil release. Br J Haematol 2005, 131:508–519.
Jonsson F, Mancardi DA, Albanesi M, Bruhns P: Neutrophils in local and
Alves CMM-MC, Carvalho IF, Lucisano Valim YM: Application of the
systemic antibody-dependent inflammatory and anaphylactic reactions.
chemiluminescence systems to evaluate the role of Fcgamma and
J Leukoc Biol 2013, 94:643–656.
complement receptors in stimulating the oxidative burst in neutrophils.
Rosetti F, Tsuboi N, Chen K, Nishi H, Ernandez T, Sethi S, Croce K, Stavrakis
Talanta 2003, 60:601–608.
G, Alcocer-Varela J, Gomez-Martin D, van Rooijen N, Kyttaris VC, Lichtman
Shah D, Aggarwal A, Bhatnagar A, Kiran R, Wanchu A: Association between
AH, Tsokos GC, Mayadas TN: Human lupus serum induces neutrophil-
T lymphocyte sub-sets apoptosis and peripheral blood mononuclear
mediated organ damage in mice that is enabled by Mac-1 deficiency.
cells oxidative stress in systemic lupus erythematosus. Free Radic Res
J Immunol 2012, 189:3714–3723.
Furebring M, Hakansson L, Venge P, Sjolin J: Differential expression of the
Fukushima K, Ando M, Ito K, Suga M, Araki S: Stimulus- and cumulative
C5a receptor and complement receptors 1 and 3 after LPS stimulation
dose-dependent inhibition of O2- production by polymorphonuclear
of neutrophils and monocytes. Scand J Immunol 2004, 60:494–499.
leukocytes of patients receiving corticosteroids. J Clin Lab Immunol 1990,
Smith CW: Leukocyte-endothelial cell interactions. Semin Hematol 1993,
30:45–53. discussion 54–45.
Nossent JC, Swaak AJ: Prevalence and significance of haematological
Bedard K, Krause KH: The NOX family of ROS-generating NADPH oxidases:
abnormalities in patients with systemic lupus erythematosus. Q J Med
physiology and pathophysiology. Physiol Rev 2007, 87:245–313.
Hansson M, Hermodsson S, Brune M, Mellqvist UH, Naredi P, Betten A,
Hepburn AL, Narat S, Mason JC: The management of peripheral blood
Gehlsen KR, Hellstrand K: Histamine protects T cells and natural killer cells
cytopenias in systemic lupus erythematosus. Rheumatology (Oxford) 2010,
against oxidative stress. J Interferon Cytokine Res 1999, 19:1135–1144.
Hansson M, Romero A, Thoren F, Hermodsson S, Hellstrand K: Activation of
Papadaki HA, Boumpas DT, Gibson FM, Jayne DR, Axford JS, Gordon-Smith
cytotoxic lymphocytes by interferon-alpha: role of oxygen radical-producing
EC, Marsh JC, Eliopoulos GD: Increased apoptosis of bone marrow CD34
mononuclear phagocytes. J Leukoc Biol 2004, 76:1207–1213.
(+) cells and impaired function of bone marrow stromal cells in patientswith systemic lupus erythematosus. Br J Haematol 2001, 115:167–174.
Holmdahl R, Sareila O, Pizzolla A, Winter S, Hagert C, Jaakkola N, Kelkka T,
Elghetany MT: Surface antigen changes during normal neutrophilic
Olsson LM, Wing K, Backdahl L: Hydrogen peroxide as an immunological
development: a critical review. Blood Cells Mol Dis 2002, 28:260–274.
transmitter regulating autoreactive T cells. Antioxid Redox Signal 2013,
Furebring M, Hakansson LD, Venge P, Nilsson B, Sjolin J: Expression of the
C5a receptor (CD88) on granulocytes and monocytes in patients with
Mellqvist UH, Hansson M, Brune M, Dahlgren C, Hermodsson S, Hellstrand K:
severe sepsis. Crit Care 2002, 6:363–370.
Natural killer cell dysfunction and apoptosis induced by chronic
Furebring M, Hakansson L, Venge P, Sjolin J: C5a, interleukin-8 and tumour
myelogenous leukemia cells: role of reactive oxygen species and
necrosis factor-alpha-induced changes in granulocyte and monocyte
regulation by histamine. Blood 2000, 96:1961–1968.
expression of complement receptors in whole blood and on isolated
Gateva V, Sandling JK, Hom G, Taylor KE, Chung SA, Sun X, Ortmann W,
leukocytes. Scand J Immunol 2006, 63:208–216.
Kosoy R, Ferreira RC, Nordmark G, Gunnarsson I, Svenungsson E, PadyukovL, Sturfelt G, Jönsen A, Bengtsson AA, Rantapää-Dahlqvist S, Baechler EC,Brown EE, Alarcón GS, Edberg JC, Ramsey-Goldman R, McGwin G Jr, Reveille
JD, Vilá LM, Kimberly RP, Manzi S, Petri MA, Lee A, Gregersen PK, et al: A
Cite this article as: Bengtsson et al.: Low production of reactive oxygenspecies in granulocytes is associated with organ damage in systemic
large-scale replication study identifies TNIP1, PRDM1, JAZF1, UHRF1BP1
lupus erythematosus. Arthritis Research & Therapy 2014 16:R120.
and IL10 as risk loci for systemic lupus erythematosus. Nat Genet 2009,41:1228–1233.
Johnston RB: Clinical aspects of chronic granulomatus disease. Curr OpinHematol 2001, 8:17–22.
Segal BH, Leto TL, Gallin JI, Malech HL, Holland SM: Genetic, biochemical,
Submit your next manuscript to BioMed Central
and clinical features of chronic granulomatous disease. Medicine
and take full advantage of:
(Baltimore) 2000, 79:170–200.
Tan EM, Cohen AS, Fries JF, Masi AT, McShane DJ, Rothfield NF, Schaller JG,
• Convenient online submission
Talal N, Winchester RJ: The 1982 revised criteria for the classification ofsystemic lupus erythematosus. Arthritis Rheum 1982, 25:1271–1277.
• Thorough peer review
Gladman DD, Ibanez D, Urowitz MB: Systemic lupus erythematosus
• No space constraints or color ﬁgure charges
disease activity index 2000. J Rheumatol 2002, 29:288–291.
Gladman D, Ginzler E, Goldsmith C, Fortin P, Liang M, Urowitz M, Bacon P,
• Immediate publication on acceptance
Bombardieri S, Hanly J, Hay E, Isenberg D, Jones J, Kalunian K, Maddison P,
• Inclusion in PubMed, CAS, Scopus and Google Scholar
Nived O, Petri M, Richter M, Sanchez-Guerrero J, Snaith M, Sturfelt G,
• Research which is freely available for redistribution
Symmons D, Zoma A: The development and initial validation of thesystemic lupus international collaborating clinics/American college of
Submit your manuscript at www.biomedcentral.com/submit
Morning Mantra Tuesday, September 06, 2011 Indian equity indices sulked in red along with global equities on Monday after sluggish US jobs data on Friday fanned fears about the health of the world's largest economy and its fallout on ongoing global recovery amidst sustained worries about the euro zone debt crisis which prompted investors for a flight of safety from Dalal
Magnus Hirsch, Markus von Fuchs and Margret Knitter SKW Schwarz Rechtsanwälte Trademarks A Global Guide SKW Schwarz:independent and business-minded The Law Firm Areas of Work SKW Schwarz is an independent German law firm. Banking and Finance We advise companies of all sizes ranging from owner-