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Increased apoptotic neutrophils and macrophages and impaired macrophage phagocytic clearance of apoptotic neutrophils in systemic lupus erythematosus.

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ARTHRITIS & RHEUMATISM
Vol. 48, No. 10, October 2003, pp 2888–2897
DOI 10.1002/art.11237
© 2003, American College of Rheumatology
Increased Apoptotic Neutrophils and Macrophages and
Impaired Macrophage Phagocytic Clearance of
Apoptotic Neutrophils in Systemic Lupus Erythematosus
Yi Ren,1 Jinling Tang,2 M. Y. Mok,1 Albert W. K. Chan,1 Adrian Wu,1 and C. S. Lau1
Objective. To evaluate whether patients with systemic lupus erythematosus (SLE) have a higher rate of
apoptosis in and secondary necrosis of polymorphonuclear neutrophils (PMNs) and macrophages compared
with controls; to compare the rate of macrophage
phagocytic clearance of apoptotic PMNs in patients with
SLE and healthy controls; to evaluate whether in vitro
PMN and macrophage apoptosis and secondary necrosis, and the ability of macrophages to phagocytose
apoptotic bodies, are correlated with lupus disease
activity; and to determine whether macrophage clearance of apoptotic bodies in patients with SLE and
normal controls is related to certain serum factors.
Methods. Thirty-six patients with SLE and 18
healthy, nonsmoking volunteers were studied. PMNs
and monocytes were isolated from fresh blood and
cultured in the presence of different sources of serum.
Apoptotic PMNs and macrophages were examined by
annexin V binding and morphology on May–Giemsa–
stained cytopreparations, at different time points. The
presence of secondary necrotic PMNs and macrophages
was verified by staining with trypan blue. Macrophage
phagocytosis of apoptotic PMNs was measured using a
coded, observer-blinded, microscopically quantified
phagocytosis assay. Cells were cultured in the presence
of serum obtained from healthy subjects or from patients with SLE.
Results. At 5 and 24 hours, the percentage of
apoptotic PMNs from patients with SLE was significantly higher than that of PMNs from healthy subjects.
At 24 and 48 hours, the percentage of secondary necrotic
PMNs from patients with SLE was also significantly
higher than the percentage of necrotic PMNs from
controls. Serum from patients with SLE accelerated the
rate of apoptosis in and secondary necrosis of PMNs
from healthy subjects. Macrophages from SLE patients
were less capable of phagocytosing apoptotic PMNs
compared with macrophages obtained from controls.
Macrophages from patients with active SLE were less
capable of phagocytosing apoptotic PMNs than were
macrophages from patients with inactive SLE, but the
difference was not statistically significant. The percentage of phagocytosis of apoptotic PMNs by macrophages
from SLE patients correlated negatively with the SLE
Disease Activity Index, serum levels of anti–doublestranded DNA, and the erythrocyte sedimentation rate,
and correlated positively with serum levels of C3, C4,
and albumin, the hemoglobin level, and the leukocyte
count. Serum from SLE patients not only significantly
increased macrophage apoptosis in cells from healthy
subjects but also remarkably down-regulated the clearance of apoptotic PMNs by macrophages from healthy
subjects. In contrast, serum from healthy subjects significantly increased phagocytosis of apoptotic PMNs by
macrophages from SLE patients.
Conclusion. The observed increase of apoptotic
PMNs and macrophages and the poor ability of macrophages from patients with SLE to phagocytose apoptotic
bodies may indicate an impaired clearance mechanism,
which may be mediated by factors in a patient’s serum.
Supported by a grant from the Committee on Research and
Conference, The University of Hong Kong, and a Research Grants
Council Competitive Earmarked Research grant from the Hong Kong
University Grants Council.
1
Yi Ren, MD, M. Y. Mok, MD, Albert W. K. Chan, BSc,
Adrian Wu, MD, C. S. Lau, MD: Queen Mary Hospital, University of
Hong Kong, Hong Kong; 2Jinling Tang, MD: Chinese University of
Hong Kong, Hong Kong.
Address correspondence and reprint requests to C. S. Lau,
MD, University Department of Medicine, Room 807 Administration
Block, Queen Mary Hospital, 102 Pokfulam Road, Hong Kong.
E-mail: cslau@hkucc.hku.hk.
Submitted for publication October 16, 2002; accepted in
revised form June 6, 2003.
Systemic lupus erythematosus (SLE) is a multisystem autoimmune disorder with unknown etiology.
2888
APOPTOTIC CELL CLEARANCE IN SLE
B cell hyperactivity with production of multiple autoantibodies is the hallmark of SLE. It is now known that
such polyclonal B cell activation is T cell–dependent and
autoantigen-driven (1). An excessive supply of autoantigens has been suggested to be secondary to increased apoptosis in SLE (2).
Apoptosis is an energy-dependent process that
leads to the programmed destruction of cells. It is tightly
regulated by the expression of cell surface molecules
such as Fas and intracellular protooncogenes including
Bcl-2 and the Bax family (3). Fas ligation leads to the
induction of apoptosis, while Bcl-2 and the Bax family
molecules provide a positive signal for survival. A possible association between SLE and Fas-mediated apoptosis and the failure of cell tolerance was suggested
when Chu et al (4) reported a defect in the expression of
Fas messenger RNA in MRL/lpr mice. This hypothesis
was later supported by the identification of the gld gene
(which was phenotypically similar to lpr) as a nonfunctional Fas ligand gene (5). In human SLE, increased
spontaneous in vitro apoptosis in cultured peripheral
blood mononuclear cells (PBMCs) has been described
(6). Nucleosomes, which are selectively produced during
apoptosis, have been shown to be a major antigen for
autoreactive T cells in SLE (7).
The morphologic features of apoptosis include
cytoplasmic blebbing, fragmentation and condensation
of intracellular chromatin, internucleosomal digestion of
DNA, and late membrane failure (8). In the late phase
of apoptosis, anionic phospholipids (phosphatidylserine
[PS] in particular), which are initially confined to the
intracellular portion of the membrane of viable cells, are
translocated to the outer surface (9). The exposure of
anionic phospholipid moieties on the cell surface of
apoptotic cells triggers the immediate engulfment of
these cells by scavenger phagocytes.
It is prudent that apoptotic bodies are effectively
cleared to avoid release of intracellular contents that
may be immunogenic, which may induce T and B cell
autoreactivity (10,11). Impairment, if any, in phagocytic
cell clearance of apoptotic bodies may therefore have a
pathogenic role in SLE. The objectives of this study
were, therefore, 1) to study the rate of apoptosis in and
secondary necrosis of polymorphonuclear neutrophils
(PMNs) and macrophages in patients with SLE and
controls; 2) to measure the ability of phagocytic macrophages to clear apoptotic bodies in patients with SLE; 3)
to evaluate whether the in vitro rate of PMN and
macrophage apoptosis and secondary necrosis and
changes in macrophage phagocytosis of apoptotic bodies
are correlated with lupus disease activity; and 4) to
2889
determine whether factors in serum from patients with
active SLE affect PMN and macrophage apoptosis and
secondary necrosis and macrophage phagocytic clearance of apoptotic bodies.
PATIENTS AND METHODS
Tissue culture materials. All chemicals were purchased from Sigma (St. Louis, MO), unless indicated otherwise. Culture medium (Iscove’s modified Dulbecco’s medium
[IMDM] and Dulbecco’s modified Eagle’s medium) and supplements (100 units/ml penicillin, 100 ␮g/ml streptomycin, 2
mM glutamine, and 10% fetal calf serum) were obtained from
Gibco (Grand Island, NY), and sterile tissue culture plasticware was obtained from Falcon Plastics (Cockeysville, MD).
Subjects. Study subjects included 36 patients with SLE
(4 men and 32 women, mean age 32.3 years [range 18–48
years]), of whom 11 had inactive disease and 25 had active
disease at the time of study. All patients met the American
College of Rheumatology classification criteria for SLE (12).
SLE disease activity was assessed using the SLE Disease
Activity Index (SLEDAI) (13). Patients were classified as
having inactive disease if the SLEDAI score was persistently
ⱕ4 for at least 4 months prior to blood sampling. Patients with
active disease had a SLEDAI score of ⱖ5 at the time of study.
Five patients, of whom 2 were newly diagnosed as
having SLE, were receiving no treatment at the time of the
study. Seventeen patients (the nonimmunosuppressive therapy
group) were receiving low-dose steroids (prednisolone, ⱕ10
mg/day) and/or hydroxychloroquine, and 14 patients (the immunosuppressive therapy group) were receiving prednisolone
at a dosage of ⬎10 mg/day and/or immunosuppressive agents.
The immunosuppressive agents taken by patients with SLE
included azathioprine (n ⫽ 11), methotrexate (n ⫽ 1), cyclosporin A (n ⫽ 1), and mycophenolate mofetil (n ⫽ 1). Table 1
shows the clinical characteristics and drug treatment of the 36
patients studied. Eighteen healthy, nonsmoking volunteers (6
men and 12 women, mean age 34 years [range 30–40 years])
were included as controls. This study was approved by The
University of Hong Kong Faculty of Medicine Ethics Committee. Written informed consent was obtained from all subjects.
Leukocytes. A 20-ml sample of venous blood was
obtained from each subject. PMNs were isolated by dextran
sedimentation and plasma–Percoll discontinuous densitygradient centrifugation and were “aged” in tissue culture in
IMDM with 10% platelet-rich plasma-derived serum in order
to undergo apoptosis, exactly as described previously (14).
Monocytes were prepared from adherent PBMCs, using standard methods, and were cultured for 5 days in IMDM with
10% autologous serum to promote maturation into macrophages.
Assessment of PMN morphology, and flow cytometric
analysis. Apoptotic PMNs were assessed by microscopic examination of cytocentrifuge preparations fixed in methanol
and stained with May–Giemsa (14). Typically, 500 counts were
made from duplicate slides of the same experiment. Binding of
fluorescein isothiocyanate–conjugated annexin V (Immunotech, Marseilles, France) was used to assess the exposure of PS.
Propidium iodide and trypan blue were used to assess plasma
2890
REN ET AL
Table 1. Clinical characteristics and drug treatment of 36 patients
with SLE*
Characteristic
Value
Sex, no. men/no. women
Age, mean (range) years
Disease duration, mean (range) years
Patients with inactive disease/patients with
active disease
SLE drug treatment at time of study
Patients with inactive disease
No treatment
Prednisolone, ⱕ10 mg/day and/or
hydroxychloroquine
Prednisolone, ⬎10 mg/day and/or
immunosuppressive agents
Patients with active disease
No treatment
Prednisolone, ⱕ10 mg/day and/or
hydroxychloroquine
Prednisolone, ⬎10 mg/day and/or
immunosuppressive agents
4/32
32.3 (18–48)
6.2 (0–12)
11/25
3
5
3
2
12
11
* Except where indicated otherwise, values are the number of patients.
Systemic lupus erythematosus (SLE) disease activity was assessed
using the SLE Disease Activity Index (SLEDAI). Inactive disease ⫽
SLEDAI score ⱕ4 for at least 4 months prior to blood sampling; active
disease ⫽ SLEDAI score ⱖ5 at the time of study. Immunosuppressive
agents included azathioprine (taken by 3 patients with inactive disease
and 8 patients with active disease), methotrexate (1 patient with active
disease), cyclosporin A (1 patient with active disease), and mycophenolate mofetil (1 patient with active disease).
membrane permeability. Labeled cells were applied to an
EPICS XL flow cytometer (Beckman Coulter, Fullerton, CA),
which automatically and simultaneously measured the fluorescence of individual cells identified by their size-dependent
light-scattering properties.
Interaction assay. A coded, observer-blinded, microscopically quantified phagocytosis assay of macrophage ingestion of apoptotic PMNs, which has been extensively described,
illustrated, and validated (14), was used in this study. Apoptotic PMNs prepared as described above were washed once in
phosphate buffered saline, suspended in IMDM and 0.5 ⫻ 106
PMNs in 50 ␮l of medium, and added to each washed well of
macrophages cultured in 96-well plates. After interaction for
30 minutes at 37°C in 5% CO2, the wells were washed in cold
(4°C) 0.9% saline to remove noningested PMNs. The macrophage monolayer was then fixed in 2% glutaraldehyde in saline
for 2 minutes, stained with myeloperoxidase, and the proportion of macrophages ingesting PMNs was counted by inverted
light microscopy. The percentage of macrophages that had
taken up apoptotic PMNs was regarded as the overall macrophage phagocytosis index. Additionally, to better assess macrophage phagocytosis activity, the percentage of macrophages
that had ingested different numbers of apoptotic PMNs was
evaluated.
Effects of serum. To determine whether serum factors
have an effect on PMNs and monocytes undergoing apoptosis,
freshly isolated PMNs and monocytes from healthy subjects
and patients with SLE were cultured and cross-cultured with
IMDM in 10% untreated serum obtained from healthy sub-
jects or patients with SLE. The rate of PMN and monocyte
apoptosis was measured at different time points. To determine
whether serum factors have an effect on phagocytosis of PMNs
undergoing apoptosis by monocyte-derived macrophages,
freshly isolated monocytes from healthy subjects and patients
with SLE were cultured and cross-cultured with IMDM in 10%
untreated serum from healthy subjects or patients with SLE for
5 days. Apoptotic PMNs were allowed to interact with day 5
macrophages for 30 minutes, and the percentage of macrophages taking up apoptotic PMNs was counted.
Statistical analysis. The Mann-Whitney U test was
used to compare the percentage of apoptotic and necrotic
PMNs and macrophages, and macrophage phagocytosis of
apoptotic PMNs, in patients with SLE (those with active or
inactive disease and those who were or were not receiving
immunosuppressive treatment) and controls. Spearman’s rank
correlation test was used to examine the relationship between
phagocytosis capacity and various characteristics of lupus
disease activity, expressed as continuous variables. All analyses
were conducted using SAS version 6.1 software (SAS Institute,
Cary, NC).
RESULTS
Apoptotic PMNs isolated from patients with SLE
and healthy subjects. PMNs from SLE patients and
controls were isolated and cultured for different periods
of time (0, 5, and 24 hours). At each time point, cells
were harvested, and apoptosis was measured. Figure 1
shows the percentage of cells cultured with autologous
serum that underwent spontaneous apoptosis at 0, 5, and
Figure 1. Spontaneous apoptosis of polymorphonuclear neutrophils
(PMNs) during culture of purified PMNs at 0, 5, and 24 hours. The
percentage of cells undergoing apoptosis was determined after morphologic examination of May–Giemsa–stained cytospin preparations.
PMNs were derived from 11 patients with inactive systemic lupus
erythematosus (SLE), 25 patients with active SLE, and 18 healthy
controls. Results are expressed as the percentage of apoptotic cells per
total PMNs. Bars show the mean and SEM. ⴱ ⫽ P ⬍ 0.05 versus
controls.
APOPTOTIC CELL CLEARANCE IN SLE
Figure 2. Percentage of secondary necrotic PMNs detected by trypan
blue assay after 24 and 48 hours of culture. PMNs were derived from
11 patients with inactive SLE, 25 patients with active SLE, and 18
healthy controls. Bars show the mean and SEM. ⴱ ⫽ P ⬍ 0.05 versus
controls. See Figure 1 for definitions.
24 hours. At time 0, there were no significant differences
in spontaneous apoptosis between PMNs from patients
with inactive (0.96 ⫾ 0.50%) or active (0.32 ⫾ 0.19%)
SLE, and PMNs from healthy controls (0.10 ⫾ 0.10%).
After 5 hours of culture, higher percentages of apoptotic
PMNs from patients with active SLE (13.93 ⫾ 2.73%)
and those with inactive SLE (8.64 ⫾ 1.54%) were
detected when compared with controls (3.25 ⫾ 1.2%).
Interestingly, the difference between the percentage of
apoptotic PMNs from patients with active SLE and
those with inactive SLE was significant (P ⬍ 0.05). The
percentage of apoptotic PMNs at 24 hours was also
determined. PMNs from patients with inactive SLE and
patients with active SLE displayed accelerated apoptosis
(71.67 ⫾ 5.46% and 71.9 ⫾ 4.04%, respectively) when
compared with healthy subjects (58.04 ⫾ 3.94%) (P ⬍
0.05). At 24 hours, there were no differences in apoptosis between PMNs from patients with inactive or active
SLE and PMNs from patients who were or were not
receiving immunosuppressive drugs. A negative correlation between the percentage of apoptotic PMNs and the
peripheral neutrophil count (r ⫽ ⫺0.595, P ⬍ 0.02) was
observed in patients with SLE.
Secondary necrotic PMNs isolated from patients
with SLE and healthy subjects. PMNs isolated from
patients with SLE and healthy subjects were cultured for
24 and 48 hours, and necrotic PMNs were detected by
trypan blue assay. At 24 hours and 48 hours, the
percentage of necrotic PMNs from patients with inactive
or active SLE was significantly higher than the percentage of necrotic PMNs from healthy subjects (Figure 2).
At both 24 and 48 hours, there were no differences in
2891
PMN necrosis between patients with active SLE and
patients with inactive SLE, and patients who were or
were not receiving immunosuppressive drugs.
Effects of serum on PMN apoptosis and necrosis.
To study whether serum from patients with SLE affected apoptosis in and necrosis of healthy PMNs, PMNs
from healthy subjects were cultured with serum from
patients with active SLE, and apoptosis was detected
(Figure 3A). Serum from patients with active SLE not only
accelerated apoptosis of PMNs from healthy subjects at 24
hours (from 58.03 ⫾ 3.94% to 72.07 ⫾ 5.17%; P ⬍ 0.05),
but also increased the percentage of necrotic PMNs at both
24 and 48 hours (Figure 3B). Serum from healthy subjects
did not rescue SLE PMNs from undergoing apoptosis and
necrosis (data not shown).
Apoptosis in macrophages from SLE patients.
Monocytes isolated from patients with SLE and healthy
Figure 3. A, Modulation of apoptosis of control PMNs by serum from
patients with SLE. PMNs from 11 healthy subjects were cultured with
serum from patients with active SLE, and apoptosis was detected at 5
hours and 24 hours. B, Modulation of necrosis in control PMNs by
serum from patients with SLE. PMNs from 11 healthy subjects were
cultured with serum from patients with active SLE, and necrosis was
detected at 24 hours and 48 hours. Bars show the mean and SEM. ⴱ ⫽
P ⬍ 0.05 versus controls. See Figure 1 for definitions.
2892
REN ET AL
from patients with SLE (Figure 5A) and healthy subjects
(Figure 5B). The percentage of SLE macrophages ingesting apoptotic PMNs was 23.48 ⫾ 7.0%, while the
percentage of healthy macrophages taking up apoptotic
PMNs was 38.5 ⫾ 4.0% (P ⬍ 0.001) (Figure 6A). Data
presented here were obtained from unselected patients.
Furthermore, the percentage of phagocytosis of apopto-
Figure 4. Apoptotic macrophages detected by flow cytometry. Monocytes isolated from 10 healthy control subjects (A) and 11 patients with
active SLE (B) were cultured with autologous serum for 5 days. The
percentage of apoptotic macrophages was determined by annexin V
binding and propidium iodide (PI) staining. The mean ⫾ SEM
percentage of annexin V binding in SLE and control macrophages was
23.25 ⫾ 3.6 and 2.02 ⫾ 0.8, respectively. The mean ⫾ SEM percentage
of PI binding in SLE and control macrophages was 14.0 ⫾ 1.6 and
2.43 ⫹ 0.5, respectively. FITC ⫽ fluorescein isothiocyanate (see Figure
1 for other definitions).
subjects were cultured for 5 days, and then apoptosis was
detected. Macrophages undergoing apoptosis were also
detected by flow cytometry (Figure 4). Macrophages
from patients with active SLE had more annexin V
binding compared with macrophages from healthy subjects. Serum from patients with active SLE accelerated
apoptosis of macrophages from healthy subjects (data
not shown).
Macrophage phagocytosis of PMNs undergoing
apoptosis. Monocytes from patients with SLE and
healthy subjects were cultured for 5 days with serum
obtained from either SLE patients or controls. Adherent
cultures of day 5 macrophages were washed and interacted with apoptotic PMNs in medium with no added
serum for 30 minutes. Figure 5 shows the results of
macrophage phagocytosis of apoptotic PMNs obtained
Figure 5. Decreased phagocytosis of apoptotic polymorphonuclear
neutrophils (PMNs) by monocyte-derived macrophages from patients
with systemic lupus erythematosus (SLE). PMNs within macrophages
are readily identifiable by their brown staining for myeloperoxidase. A,
Macrophages from patients with active SLE ingesting apoptotic PMNs
(day 5). B, Macrophages from healthy subjects ingesting apoptotic
PMNs (day 5).
APOPTOTIC CELL CLEARANCE IN SLE
2893
Figure 7. Ingestion of apoptotic PMNs by macrophages from healthy
subjects and patients with SLE. Day 5 macrophages from 8 SLE
patients and 8 healthy subjects were allowed to interact with apoptotic
PMNs for 30 minutes. The number of macrophages that had ingested
different numbers of apoptotic PMNs was counted. Bars show the
mean and SEM. See Figure 5 for definitions.
Figure 6. Macrophage ingestion of apoptotic PMNs from healthy
subjects and patients with SLE. Day 5 macrophages from SLE patients
and healthy subjects (n ⫽ 12) were allowed to interact with apoptotic
PMNs for 30 minutes. The number of macrophages ingesting apoptotic
PMNs was then counted. A, Phagocytosis of apoptotic PMNs by
macrophages from unselected SLE patients (n ⫽ 36). ⴱ ⫽ P ⬍ 0.001
versus controls. B, Phagocytosis of apoptotic PMNs by macrophages
from patients with active (n ⫽ 25) or inactive (n ⫽ 11) SLE and healthy
control subjects. ⴱ ⫽ P ⬍ 0.05 versus controls; ⴱⴱ ⫽ P ⬍ 0.001 versus
controls. Bars show the mean and SEM. See Figure 5 for definitions.
tic PMNs by macrophages from patients with inactive or
active SLE was 29.2 ⫾ 5.2% and 17.8 ⫾ 3.2%, respectively (Figure 6B); this difference was not statistically
significant (P ⫽ 0.081). The use of immunosuppressive
agents did not appear to influence macrophage phagocytosis of apoptotic PMNs. There were no significant
differences in phagocytosis between healthy subjects and
patients with inactive SLE.
Not only did patients with SLE have fewer macrophages that were capable of phagocytosing apoptotic
PMNs, but also the capacity of the phagocytosiscompetent macrophages to ingest apoptotic bodies was
lower in SLE patients compared with controls. Figure 7
shows the percentage of macrophages that ingested
different numbers of apoptotic PMNs from patients with
SLE and controls. Figure 8 shows the number of apo-
ptotic PMNs within 100 phagocytosis-competent macrophages, as well as within a total of 100 macrophages
(both phagocytosis-competent and phagocytosisnoncompetent) from patients with SLE and controls.
Correlation between the percentage of phagocytosis of apoptotic PMNs by SLE macrophages and lupus
disease activity parameters. The percentage of phagocytosis of apoptotic PMNs by macrophages from patients with SLE correlated negatively with the SLEDAI
score, the serum level of anti–double-stranded DNA
Figure 8. Impaired phagocytic capacity of SLE macrophages. The
number of apoptotic PMNs (from patients with SLE [n ⫽ 8] and
controls [n ⫽ 8]) within 100 phagocytically competent macrophages,
and within a total of 100 macrophages (both phagocytically competent
and phagocytically noncompetent) was counted. Bars show the mean
and SEM. ⴱ ⫽ P ⬍ 0.05 versus controls; ⴱⴱ ⫽ P ⬍ 0.001 versus
controls. See Figure 5 for definitions.
2894
REN ET AL
Table 2. Correlation between macrophage phagocytosis of apoptotic
neutrophils and various parameters of SLE disease activity*
Parameter
r
P
SLEDAI
Anti-dsDNA
C3
C4
Serum albumin
Hemoglobin
Leukocyte count
Erythrocyte sedimentation rate
⫺0.5169
⫺0.7184
0.5011
0.5556
0.6058
0.5330
0.5181
⫺0.5168
0.0336
0.0017
0.0480
0.0254
0.0129
0.0335
0.0335
0.0404
* SLE ⫽ systemic lupus erythematosus; SLEDAI ⫽ SLE Disease
Activity Index; anti-dsDNA ⫽ anti–double-stranded DNA.
increased in SLE (19). In that study, there was a positive
correlation with anti-dsDNA antibodies and lupus disease activity, as measured by the Systemic Lupus Activity Measure score (20).
It is interesting to note from our experiments that
serum from patients with active SLE accelerated in vitro
apoptosis in and secondary necrosis of PMNs, while
serum from control subjects failed to prevent cells from
SLE patients from undergoing apoptosis and secondary
necrosis. These results suggest that both genetic and
serum factors that regulate PMN apoptosis may be
abnormal in patients with SLE. Apoptosis in PMNs is
mediated by Fas, and Fas ligation is associated with
widespread PS expression on the cell surface (21).
(anti-dsDNA), and the erythrocyte sedimentation rate,
and positively with the serum levels of C3, C4, and
albumin, the hemoglobin level, and the leukocyte count
(Table 2).
Effects of serum on phagocytosis of apoptotic
PMNs. We also studied whether serum from healthy
subjects would rescue the impaired phagocytic capacity
of macrophages obtained from patients with active SLE.
Monocytes from patients with SLE were cultured for 5
days with serum obtained from healthy subjects and then
interacted with apoptotic PMNs for 30 minutes. Healthy
serum increased the percentage of phagocytosis of apoptotic PMNs by macrophages from patients with active
SLE, from 23.48 ⫾ 7.0% to 52.92 ⫾ 6.9% (P ⬍ 0.05)
(Figure 9A). In contrast, serum obtained from patients
with active SLE decreased the percentage of macrophages ingesting apoptotic PMNs, from 41.4 ⫾ 5.3% to
14.0 ⫾ 2.3% (P ⬍ 0.05) (Figure 9B).
DISCUSSION
In this study, we observed that the rate of in vitro
apoptosis of PMNs and macrophages was increased in
patients with SLE when compared with controls. Patients with active disease had higher rates of in vitro
apoptosis than did patients with inactive disease. Additionally, the in vitro rate of secondary necrosis of PMNs
was increased in patients with SLE when compared with
controls. Our findings are in accordance with previous
studies, in which it was reported that increased apoptosis
of peripheral blood cells has a pathogenic role in SLE
(6,15–19). Most of these studies evaluated the increased
spontaneous cell death of PBMCs in SLE; the rate of
lymphocyte apoptosis correlated significantly with disease activity (6,15,17). Recently, neutrophil apoptosis, as
assessed by flow cytometry using annexin V binding and
fluorescence-labeled anti-Fas, was also shown to be
Figure 9. Effects of serum on phagocytosis of apoptotic polymorphonuclear neutrophils (PMNs). A, Monocytes isolated from patients with
active systemic lupus erythematosus (SLE) (n ⫽ 6) were cultured with
autologous serum and serum from healthy subjects. The number of
macrophages ingesting apoptotic PMNs was counted. B, Monocytes
isolated from healthy subjects (n ⫽ 6) were cultured with autologous
serum and serum from patients with active SLE. Bars show the mean
and SEM. ⴱ ⫽ P ⬍ 0.05.
APOPTOTIC CELL CLEARANCE IN SLE
Significantly increased PMN Fas expression in SLE has
been reported (19).
Besides being mediated by Fas expression, PMN
apoptosis is regulated by a number of serum-soluble
factors, primarily cytokines, including interleukin-1␤
(IL-1␤), IL-4, IL-6, granulocyte–macrophage colonystimulating factor (GM-CSF), and granulocyte colonystimulating factor, which delay apoptosis, and tumor
necrosis factor ␣ (TNF␣), which induces apoptosis
(22,23). Cytokine abnormalities have been reported in
SLE (24). It is possible that the presence, or the lack, of
some of these PMN apoptosis regulatory cytokines in
the serum of patients with active SLE accelerates control
of PMN apoptosis and secondary necrosis. This possibility should be further investigated. We previously
examined the question of whether allospecific antibodies
may have an effect on PMN apoptosis and necrosis, by
performing cross-feeding experiments in which normal
cells were incubated with sera from different healthy
individuals. No differences in the rate of PMN apoptosis
and necrosis were observed (Ren Y, et al: unpublished
observations).
Whether the use of steroids and other immunosuppressive drugs in the treatment of SLE may account
for these observations was not demonstrated in the
current study, because only a few patients were not
receiving any medication at the time of the study. This is
an inherent problem with most human SLE studies,
because few patients are treatment naive. We did,
however, evaluate whether the use of high-dose steroids
(prednisolone, ⬎10 mg/day) and/or immunosuppressive
drugs may influence our results. There were no significant differences in the rate of apoptosis and macrophage
handling of apoptotic cells between patients in the
immunosuppressive therapy group and those in the
nonimmunosuppressive therapy group. This is probably
related to heterogeneity of the clinical characteristics,
disease activity, steroid dose, and use of different immunosuppressive drugs within the 2 treatment groups,
which may confound the analysis.
In SLE, an increased number of circulating
PMNs is probably more important than is PBMC apoptosis, because PMNs are numerically greater and have
a shorter lifespan compared with PBMCs. Increased
apoptotic PMNs may contribute to an excess of autoantigens, including dsDNA and nucleosomes. Nucleosome
release has recently been shown to be important in both
the induction of SLE and the initiation of renal lesions
by targeting autoantibodies to the glomerular basement
membrane (25). The relationship between increased
apoptosis, nucleosome release, and the formation of
2895
nucleosome–immunoglobulin complexes was confirmed
by Licht et al (26), using lipopolysaccharide (LPS)
administration to induce apoptosis in the MRL lupusprone model.
A fast and efficient way to remove cells undergoing apoptosis and secondary necrosis is important for
preventing the release of unwanted proteins that may
accumulate within the lymphatic system and ultimately
trigger antinuclear immune responses (11). In vivo, cells
undergoing apoptosis are usually recognized and swiftly
ingested by macrophages or neighboring cells acting as
semiprofessional phagocytes. The phagocytes then actively down-regulate inflammatory and immune responses, further ensuring the safe clearance of apoptotic
cells in vivo. The reduced phagocytic activity of PMNs,
monocytes, and macrophages in patients with SLE is
well established (27–29), and aberrations in the clearance of apoptotic materials by phagocytes have been
hypothesized to play a role in the etiology of lupus
(19,30).
Another important finding of this study was that
macrophages from patients with SLE were less capable
of ingesting apoptotic PMNs when compared with macrophages from control subjects. This is in concordance
with the finding by Herrmann et al (31) of decreased
noninflammatory engulfment phagocytosis of apoptotic
PBMCs. We went one step further, because our study is
the first to show that the ability of macrophages to
phagocytose apoptotic cells is inversely correlated with
biochemical and serologic markers of lupus disease
activity and the overall SLEDAI score. Additionally,
these findings confirmed that impaired macrophage
phagocytosis has a contributory role in the pathogenesis
of SLE.
The underlying mechanism of defective macrophage phagocytosis in SLE has not been fully evaluated.
In cross-feeding experiments, Herrmann et al (31)
showed that reduced clearance of apoptotic cells in SLE
was not related to an “abnormal execution of apoptosis,”
but rather to intrinsic phagocyte dysfunction in patients
with SLE. In our study, serum obtained from patients
with active SLE impaired the phagocytic ability of
healthy control macrophages. Conversely, serum from
control subjects restored the phagocytic ability of macrophages from patients with SLE. These data suggest that
in patients with SLE, the impaired ability of macrophages to phagocytose apoptotic PMNs is secondary to
the presence or absence of certain serum factors rather
than being attributable to an intrinsic defect of the
macrophage or its binding to apoptotic PMNs.
The identity of the serum factor(s) that influ-
2896
ences macrophage phagocytosis of apoptotic cells is not
known. Several recognition molecules or phagocyte receptors have been identified. These include lectins, ␣v␤3
(vitronectin receptor), thrombospondin (TSP), CD36
(TSP receptor), PS receptors, scavenger receptors,
CD14 (the LPS receptor), ATP-binding cassette protein
1, and complement factors (11). One or more of these
molecules may be lacking or may meet with interference
in SLE. Cairns et al (32) also recently demonstrated
reduced expression of CD44 on PMNs, which may result
in poor recognition and clearance of these cells by
monocyte-derived macrophages.
A deficiency of complement and complement
receptors confers an increased risk of SLE (33). We
previously reported that insufficiency of mannosebinding lectin (MBL), a newly derived complementactivating factor, is associated with the development of
SLE in Chinese individuals of southern origin (34,35).
C1q and other complement factors have been shown to
bind to surface blebs of apoptotic cells and facilitate
phagocytic removal (36,37). Recently, MBL was shown
to have similar properties (38). Thus, impairment in
serum complement and/or MBL may render reduced
clearance of apoptotic PMNs. It should be noted that in
our study, the macrophage phagocytosis index correlated positively and significantly with serum levels of C3
and C4; serum MBL levels were not measured.
Antiphospholipid antibodies (aPL), which are
present in 20–40% of patients with SLE, may also
disturb the safe clearance of apoptotic cells by macrophages that do not bear opsonic immunoglobulin (11).
Alternatively, aPL may opsonize apoptotic cells so that
macrophages incite inflammation (e.g., release of
TNF␣) or autoimmunity (39). In our study, we evaluated
the relationship between macrophage phagocytosis ability and serum levels of IgG and IgM anticardiolipin
antibodies. However, no significant correlation was
found (data not shown).
The effects of other autoantibodies on macrophage apoptotic body phagocytosis have not been thoroughly studied, but it is tempting to speculate that
anti-dsDNA antibodies, which cross-react with antinucleosome antibodies, may have a role. Indeed, nucleosomes have been shown to elicit a strong dosedependent inhibition of phagocytosis of apoptotic
thymocytes by young macrophages from MRL mice (40).
Antineutrophil cytoplasmic antibodies (ANCAs) have
also been shown to induce the generation of reactive
oxygen species and cause dysregulation of primed PMN
apoptosis and clearance by macrophages (41). However,
ANCAs are not commonly found in patients with SLE.
REN ET AL
Finally, it is possible that the presence of allospecific
antibodies in a patient’s serum may interfere with the
ability of macrophages to remove apoptotic cells. However, this possibility has not been studied previously.
Proinflammatory cytokines have been shown to
potentiate TSP-mediated phagocytosis of PMNs undergoing apoptosis (10). For example GM-CSF, IL-1␤,
TNF␣, interferon-␣, and transforming growth factor ␤1
enhance phagocytosis, while IL-4 and IL-6 have no
effects. As was mentioned above, SLE is associated with
widespread changes in cytokine levels (24). It is possible
that lowered levels of some of these proinflammatory
cytokines may explain the results of our study.
In conclusion, results of our study confirm previous findings of increased peripheral blood cell apoptosis
that correlated with increased disease activity in SLE. To
prevent induction of autoimmunity, it is prudent that
apoptotic cells are effectively removed, and the most
important mechanism for such removal is nonphlogistic
phagocytic clearance by macrophages. Our data confirm
that this mechanism is defective in SLE. Additionally,
impairment in macrophage apoptotic cell clearance correlated with SLE disease activity. This further supports
an important role of such a defect in the etiology of SLE.
The reason why macrophages from patients with SLE
are ineffective in engulfing apoptotic cells is not fully
understood, but our results suggest that this is likely due
to the presence or the lack of a specific serum factor(s)
in these patients. Further studies in this area are required.
ACKNOWLEDGEMENTS
We thank Ms Y. Lo for her assistance in the collection
of SLE clinical data and Ms K. Larm for secretarial help.
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