Remnants of Secondarily Necrotic Cells Fuel Inflammation inSystemic Lupus Erythematosus
Luis E. Munoz,1 Christina Janko,1 Gerhard E. Grossmayer,1 Benjamin Frey,1
Reinhard E. Voll,1 Peter Kern,2 Joachim R. Kalden,1 Georg Schett,1 Rainer Fietkau,1
Martin Herrmann,1 and Udo S. Gaipl1
Objective. Patients with systemic lupus erythem-atosus (SLE) are often characterized by cellular as wellas humoral deficiencies in the recognition and phago-cytosis of dead and dying cells. The aim of this study wasto investigate whether the remnants of apoptotic cellsare involved in the induction of inflammatory cytokinesin blood-borne phagocytes.
Methods. We used ex vivo phagocytosis assayscomprising cellular and humoral components andphagocytosis assays with isolated granulocytes andmonocytes to study the phagocytosis of secondarilynecrotic cell–derived material (SNEC). Cytokines weremeasured by multiplex bead array technology.
Results. We confirmed the impaired uptake ofvarious particulate targets, including immunoglobulin-opsonized beads, by granulocytes and monocytes frompatients with SLE compared with healthy control sub-jects. Surprisingly, blood-borne phagocytes from two-thirds of the patients with SLE took up SNEC, which
was rarely phagocytosed by phagocytes from healthycontrol subjects or patients with rheumatoid arthritis.Supplementation of healthy donor blood with IgG frac-tions derived from patients with SLE transferred thecapability to take up SNEC to the phagocytes of healthydonors. Phagocytosis-promoting immune globulins alsoinduced secretion of huge amounts of cytokines byblood-borne phagocytes following uptake of SNEC.
Conclusion. Opsonization of SNEC by autoanti-bodies from patients with SLE fosters its uptake byblood-borne monocytes and granulocytes. Autoantibody-mediated phagocytosis of SNEC is accompanied bysecretion of inflammatory cytokines, fueling the inflam-mation that contributes to the perpetuation of autoim-munity in SLE.
Systemic lupus erythematosus (SLE) is a multi-factorial disease characterized by a chronic and antigen-driven autoimmune response against antigens that areoften cleaved, otherwise modified, or translocated indying or dead cells (1). In a subgroup of patients, analteration in the recognition and clearance of apoptoticcells by phagocytes is thought to contribute to pathogen-esis. In healthy individuals, dying cells are cleared at veryearly stages of the death program, eliciting neitherinflammation nor an immune response. Instead, apopto-tic cells induce immunosuppressive effects (2). If theyare not promptly cleared, they may undergo secondarynecrosis, releasing modified (nuclear) autoantigens andendogenous “danger signals,” such as high mobilitygroup box protein 1, uric acid, and ATP, that fosterinflammation. In patients with SLE, deficient clearanceof apoptotic cells induces the accumulation in varioustissues of nuclear debris containing a plethora of auto-antigens. In germinal centers, these autoantigens mayserve as selecting antigen, providing survival signals forB cells that accidentally became autoreactive during the
Supported by the DFG, SFB 643 (project B5), the Interdis-ciplinary Center for Clinical Research (IZKF N2) at the ErlangenUniversity Hospital, the European Commission (European UnionQLK3-CT-2002-02017_APOCLEAR), and the Lupus Erythema-thodes Selbsthilfegemeinschaft e.V. Dr. Munoz’ work was supportedby a Programme AlBan scholarship (E04D047956VE) from the Euro-pean Union Program of High Level Scholarships for Latin America.
1Luis E. Munoz, MD, MSc, Christina Janko, Dipl Biol,Gerhard E. Grossmayer, MD, Benjamin Frey, PhD, MSc, Reinhard E.Voll, MD, Joachim R. Kalden, MD, Georg Schett, MD, RainerFietkau, MD, Martin Herrmann, PhD, Udo S. Gaipl, PhD: UniversityHospital of Erlangen, Friedrich-Alexander University of Erlangen–Nuremberg, Erlangen, Germany; 2Peter Kern, MD: Franz von Prum-mer Hospital for Rheumatology and General Hospital, Bad Bruck-enau, Germany.
Drs. Herrmann and Gaipl contributed equally to this work.Address correspondence and reprint requests to Udo S.
Gaipl, PhD, Department of Radiation Oncology, University Hospitalof Erlangen, Friedrich-Alexander University of Erlangen–Nuremberg,Universitaetsstrasse 27, 91054 Erlangen, Germany. E-mail: [email protected].
Submitted for publication August 14, 2008; accepted inrevised form February 20, 2009.
1733
process of somatic mutation (3). Thus, apoptotic cell–derived antigens contribute to the etiology of auto-immunity in patients with SLE.
Clearance deficiencies and elevated apoptosisrates in patients with SLE may be the cause of theincreased levels of circulating nuclear material (4),which in this report is referred to as secondarily necroticcell–derived material (SNEC). SNEC is considered toform immune complexes with nuclear autoantibodiesand to induce tissue damage after deposition and/orlocal activation of inflammatory responses (5). Nucleicacid–containing immune complexes from patients withSLE induced type I interferon (IFN) production uponinteraction with plasmacytoid dendritic cells, which playan important role in pathogenesis (6). The effect ofSNEC and its complexes in the circulation has not yetbeen investigated.
Granulocytes and monocytes are circulatingphagocytes that play an important role in the defenseagainst pathogens. We hypothesized that granulocytesand monocytes also participate in the inflammatoryreactions characteristic of SLE, after interaction withSNEC generated from apoptotic cells that were notproperly cleared. It has long been recognized that iso-lated granulocytes and macrophages from patients withSLE show defects in the in vitro phagocytosis of yeastand IgG-opsonized erythrocytes (7).
Autoantibodies against nuclear antigens of theIgG isotype are a hallmark of SLE (8). Frisoni et alpostulated that these autoantibodies may shift dyingcells from regular clearance by macrophages to engulf-ment by immature dendritic cells (9). This may causeinflammatory clearance and perpetuation of the auto-immune response (9), probably mediated by type I IFNs(10). Immune complex deposition or in situ formation intissues induces chemotaxis and infiltration of inflamma-tory cells, which are accelerated by complement activa-tion. After stimulation with ultraviolet B (UVB) light,accumulation of apoptotic cell–derived material in theskin of patients with cutaneous lupus (11) or SLE hasbeen described to induce inflammatory infiltrates (12).This suggests that circulating, deposited, or in situ–generated SNEC in free or in complexed form may baitblood-borne phagocytes to participate in the chronifica-tion of the inflammatory response of SLE.
PATIENTS AND METHODS
Patients. All participants in the study were patients atour outpatient clinic and met the diagnostic criteria of theAmerican College of Rheumatology (13). Sex- and age-
matched normal healthy donors served as a control population.Informed consent was obtained from all blood donors, and thestudy received final approval from the ethics committee ofErlangen University Hospital. Heparinized venous wholeblood was collected by venipuncture and processed within 2hours of collection. Plasma and serum samples were obtainedby centrifugation at 1,000g for 15 minutes of heparinized andclotted blood, respectively, stored at �20°C, and thawed oncefor further analysis. Immunoglobulin fractions were preparedfrom the sera of normal healthy donors and patients with SLEby direct ammonium sulfate precipitation (final concentration45%) followed by dialysis against phosphate buffered saline(PBS). Antibody depletion was achieved by affinity chroma-tography with double-stranded DNA (dsDNA)–cellulose(Sigma, Munich, Germany). The serologic parameters ob-tained from our routine laboratory, the European ConsensusLupus Activity Measurement index, treatment, and organinvolvement of each SLE patient are summarized in Table 1.
Particulate targets for the phagocytosis assays. Flu-oresbrite YG beads (1 �m; Polysciences, Warrington, PA)were coated with 10 �g/ml human immunoglobulin (pentaglo-bin; Biotest, Vienna, Austria) by passive adsorption for 2 hoursin 0.1M carbonate buffer. Zymosan A (derived from Saccha-romyces cerevisiae)–BioParticles–fluorescein conjugate andacetylated low-density lipoprotein (LDL) conjugated with Al-exa Fluor 488 were obtained from Invitrogen (Karlsruhe,Germany). Green fluorescent protein–expressing recombinantEscherichia coli (DH5�) was kindly provided by Dr. MichaelHensel (Institute for Clinical Microbiology, University ofErlangen–Nuremberg).
SNEC can be generated by various methods, as fol-lows: 1) digestion by serum DNase I and C1q of humanlymphocytes that were treated with heat (56°C), as describedpreviously (14), 2) UVB irradiation of human lymphocytes thatthen progress to secondary necrosis, and 3) UVB irradiation ofhuman lymphocytes followed by heat treatment in the earlystages of apoptosis. Particles generated by any of these meth-ods show similar DNA content, scatter, and ligand-bindingcharacteristics, as detected by flow cytometry (data notshown). In order to use identical material for all patients andcontrol subjects, we had to freeze the prey, which was mostpossible using SNEC generated by treatment (method 1).Briefly, necrosis was induced by treatment of viable cells at56°C for 30 minutes. SNEC was then obtained by consecutiveincubation at 37°C in standard medium containing 5% humanAB serum. SNEC was stained with 10 �g/ml propidium iodide(PI; Sigma) and stored at �20°C until used.
We further characterized SNEC by analyzing the bind-ing to SNEC of the following fluorescence-labeled dying anddead cell ligands and control proteins: bovine serum albumin(BSA), human IgG, human fetuin, Narcissus pseudonarcissuslectin, acetylated LDL, chicken annexin V, human C-reactiveprotein (CRP), and complement C1q as well as C3c. Thestaining of SNEC was carried out in PBS/EDTA or in Ringer’ssolution, and binding of the ligands was quantified by flowcytometry.
Whole blood phagocytosis assays. Freshly drawn bloodwas mixed at 4°C with immunoglobulin-opsonized beads (4 �107 particles/ml) or SNEC (1 � 105 nuclei/ml). Other partic-ulate targets such as albumin-coated beads, zymosan, bacteria,and acetylated LDL were also tested. One hundred microliters
1734 MUNOZ ET AL
Tab
le1.
Sero
logi
can
dcl
inic
alfe
atur
esof
the
patie
nts
with
SLE
*
Patie
ntA
ge,
year
sSe
xD
iagn
osis
,ye
ars
EC
LA
ME
SR,
mm
/hou
rC
RP,
mg/
dlC
3,m
g/dl
C4,
mg/
dlA
nti-d
sDN
A,
units
/ml
AN
A,
titer
Org
anin
volv
emen
tT
reat
men
t
Join
tsB
lood
Sero
sal
mem
bran
esSk
inK
idne
yC
NS
Pred
niso
ne,
mg/
day
CQ
MT
XA
ZA
157
M0
5N
D0.
111
714
.68
0X
XX
15X
264
F23
28
1.5
126
215.
93,
200
X5
328
F6
3N
DN
D95
8.7
14.5
3,20
0X
X5
464
F4
1N
D0.
411
119
.20.
132
0X
100
522
F3
227
113
621
.54.
11,
000
X10
666
F9
27
0.4
133
20.6
0.1
1,00
0X
X5
X7
57F
173
90.
592
10.9
9.3
100
X5
X8
72F
70
140.
698
220.
13,
200
XX
5X
936
M9
717
0.4
441
629
10,0
00X
X5
X10
52F
131
ND
0.9
8017
.911
.332
0X
XX
5X
X11
30F
03
ND
0.8
154
194.
30
XX
10X
1245
F2
1N
D0.
374
105.
80
X13
46M
162
31N
D11
625
.80.
11,
000
XX
5X
1434
F2
35
0.1
7915
0.1
0X
7.5
XX
1563
F23
ND
130.
312
315
.30.
11,
000
1653
F2
321
0.5
102
20.5
35.4
320
XX
X5
X17
26M
70
20.
199
19.3
0.1
0X
XX
5X
X18
46F
162
230.
586
1712
81,
000
XX
5X
X19
41F
143
90.
155
10.9
23.1
1,00
0X
XX
10X
2030
F1
213
1.1
142
260.
10
XX
10X
2167
F26
26
111
512
.89.
41,
000
X2.
522
65F
261
ND
0.8
109
2811
1,00
0X
XX
XX
X23
22F
15
241.
945
1.9
6932
0X
XX
XX
4X
2430
F6
115
0.1
748.
727
100
X5
XX
2524
F4
010
0.4
8918
.623
.910
0X
XX
2646
F13
213
0.4
105
110.
11,
000
XX
2766
FN
D1
320.
610
122
.30.
11,
000
XX
2828
F5
422
0.1
8411
0.1
100
XX
5X
2967
F16
17
1.1
786.
40.
11,
000
X2
X30
71F
92
442.
211
021
.93.
81,
000
XX
XX
3127
F0
311
0.2
8513
.40.
11,
000
XX
X5
XX
3239
FN
D3
520.
388
13.1
31.1
320
XX
X33
29M
ND
311
0.3
9212
.323
.910
0X
7.5
XX
3464
F17
219
0.1
110
147.
80
XX
X35
64F
ND
114
0.3
111
197.
83,
200
XX
5X
3622
F9
14
0.2
7410
.914
.81,
000
XX
5X
3758
F8
1N
D0.
410
919
.15.
732
0X
XX
3834
F12
319
0.1
7317
.85.
91,
000
XX
3937
F5
4N
D0.
176
133.
610
0X
5X
4023
F3
6N
D0.
143
1.7
16.1
1,00
0X
XX
4152
F6
327
0.4
100
12.4
0.1
100
XX
5X
4249
M7
221
0.3
ND
ND
4.9
1,00
0X
X7.
543
24F
16
100
0.6
8611
365
1,00
0X
2044
71F
85
230.
510
915
.712
.71,
000
XX
4X
4541
F7
3N
D0.
183
11.4
6.2
320
X5
4631
FN
D4
ND
0.1
6917
.650
.450
0X
X7.
547
34F
34
ND
0.1
6210
4.7
320
XX
7.5
X48
45F
173
150.
389
220.
10
X10
XX
4945
M12
ND
ND
0.3
9213
.521
1,00
0X
4X
5071
F20
ND
ND
0.6
110
19.4
0.1
0X
7.5
5133
F1
643
0.1
107
2877
.532
0X
X15
*N
orm
alva
lues
are
asfo
llow
s:fo
rC
-rea
ctiv
epr
otei
n(C
RP)
,�0.
8m
g/dl
;for
C3,
79–1
52m
g/dl
;for
C4,
16–4
7m
g/dl
;for
anti–
doub
le-s
tran
ded
DN
A(a
nti-d
sDN
A),
�7
units
/ml;
for
antin
ucle
aran
tibod
ies
(AN
A),
�1:
100.
SLE
�sy
stem
iclu
pus
eryt
hem
atos
us;
EC
LA
M�
Eur
opea
nC
onse
nsus
Lup
usA
ctiv
ity
Mea
sure
men
tin
dex;
ESR
�er
ythr
ocyt
ese
dim
enta
tion
rate
;CN
S�
cent
raln
ervo
ussy
stem
;CQ
�ch
loro
quin
e;M
TX
�m
etho
trex
ate;
AZ
A�
azat
hiop
rine
;ND
�no
tde
term
ined
.
INFLAMMATORY CLEARANCE OF SECONDARILY NECROTIC CELLS IN SLE 1735
of each mixture was incubated at 37°C and 4°C. The incubationtime was optimized at 30 minutes and 240 minutes forimmunoglobulin-opsonized beads and SNEC, respectively. Af-terward, samples were fixed using the Coulter Multi-Q-Prepsystem (Coulter Electronics, Miami, FL) and analyzed by flowcytometry (Beckman Coulter, Miami, FL). Phagocytosis indi-ces were calculated as the product of the percentage ofphagocytes and the mean fluorescence intensity of the phago-cytes.
Phagocytosis assays with isolated cells. Peripheralblood mononuclear cells (PBMCs) were isolated by Ficolldensity-gradient centrifugation. Residual platelets were re-moved by density-gradient centrifugation through a cushion offetal calf serum (Invitrogen). Monocytes were obtained bynegative selection with magnetic cell sorting (Miltenyi Biotech,Bergisch Gladbach, Germany). Granulocytes were collectedfrom the thin layer immediately above the erythrocytes afterFicoll density-gradient centrifugation. The purity of the ob-tained monocyte and granulocyte populations was �98%, asdetermined by CD14 or CD16 staining, respectively. Afterisolation, cells were cultured in the presence of SNEC incomplete medium supplemented with 30% of the respectivesera for 4 hours or 18 hours at 37°C. Supernatants werecollected for the cytokine analyses. Cells cultured in theabsence of SNEC were used as control.
Cytokine measurement. Supernatants of whole bloodand isolated phagocyte cultures from patients with SLE wereassayed for the cytokines interleukin-8 (IL-8), tumor necrosisfactor � (TNF�), TNF�, IL-6, IFN�, IL-1�, IL-18, and IL-10after 4 hours and 18 hours in the presence of SNEC, bymultiplex bead array technology (Bender MedSystems, Vi-enna, Austria). Cytokine production was recorded as thedifference between supernatants in the presence or absence ofSNEC.
Statistical analysis. Correlations were assessed using abivariate nonparametric correlation test (Spearman’s rho).Due to the heterogeneity of SLE disease, we elected not tocompare groups using the parametric Student’s t-test. Instead,we used the Kruskal-Wallis test to compare medians.
RESULTS
Binding of dying and dead cell–related ligands bySNEC. The binding of fluorescence-labeled BSA, hu-man IgG, human fetuin, N pseudonarcissus lectin, acetyl-ated LDL, chicken annexin V, human CRP, C1q, andC3c to SNEC was analyzed using flow cytometry. SNECshowed low binding of C3c and high binding of acetyl-ated LDL, annexin V, C1q, and monomeric as well aspentameric CRP (Figure 1A). The binding of theseproteins is characteristic for late apoptotic and second-arily necrotic cells.
Phagocytosis of SNEC even in SLE patients withimpaired clearance of other particulate targets. Bothcellular and humoral mechanisms are important for thesilent clearance of dying and dead cells. Therefore, weestablished an ex vivo phagocytosis assay in whole blood.
Heparinized fresh whole blood was incubated withfluorescence-labeled immunoglobulin-opsonized beadsand PI-labeled SNEC at 4°C and 37°C, respectively.After lysis of erythrocytes, the monocyte, granulocyte,and lymphocyte populations could clearly be distin-guished by flow cytometry, using their scatter properties
Figure 1. Alteration of phagocytosis of secondarily necrotic cell–derived material (SNEC) in the whole blood of patients with systemiclupus erythematosus (SLE). A, Characterization of SNEC by bindingof acetylated low-density lipoprotein (AcLDL), annexin V, monomericC-reactive protein (CRPm), C1q, C3c, and pentameric CRP (CRPp).Whole blood was cultured with fluorescent particulate prey at 4°C or37°C and analyzed by flow cytometry. B and E, Identification ofmonocytes, granulocytes, lymphocytes, and prey by their scatter prop-erties. C and D, Uptake of immunoglobulin-opsonized beads bygranulocytes. F and G, Uptake of fluorescent SNEC by granulocytes.Note that granulocytes from a patient with SLE show clearly reducedengulfment of immunoglobulin-opsonized beads compared with thosefrom a normal healthy donor (NHD). In contrast to granulocytes fromhealthy donors, those from patients with SLE were able to take upSNEC. The healthy donor granulocyte shown in D has ingestedimmunoglobulin-opsonized green fluorescent beads. The SLE granu-locyte shown in G contains red fluorescent SNEC. Bar � 5 �m. BSA �bovine serum albumin; FITC � fluorescein isothiocyanate; Npn �Narcissus pseudonarcissus lectin; MFI � mean fluorescence intensity;IS � inactive serum; AS � active serum; PI � propidium iodide;WL � white light; DAPI � 4�,6-diamidino-2-phenylindole.
1736 MUNOZ ET AL
(Figures 1B and E). The histogram in Figure 1C and theimage in Figure 1D exemplarily show the uptake ofimmunoglobulin-opsonized beads by the granulocytes ofa healthy donor and a patient with SLE. Binding andengulfment of the beads were detected at 4°C (greyareas) and 37°C (black lines), respectively, becausecytoskeleton proteins cannot effectively reorganize forthe engulfment process at low temperatures. Phagocyto-sis was then calculated as the difference between thephagocytosis indices of cells cultured at 37°C and thosecultured at 4°C. Figure 1C illustrates the impairedphagocytosis of the immunoglobulin-opsonized beads ofa patient with SLE. In striking contrast, SNEC was takenup by granulocytes from a patient with SLE (Figures 1Fand G).
In this way, we also analyzed the phagocyticcapabilities of granulocytes and monocytes in wholeblood of a cohort of patients with SLE or rheuma-toid arthritis (RA) and normal healthy donors. Granu-locytes and monocytes from patients with SLE showeda significant reduction (P � 0.05) in the uptake ofimmunoglobulin-opsonized beads (Figure 2A). A few
patients with RA also showed a similar phagocyticweakness, but to a much lesser extent.
SNEC was taken up by at least 50% of thegranulocytes and monocytes from patients with SLE(Figure 2B). Very few normal healthy donors and nopatient with RA engulfed relevant amounts of SNEC(P � 0.01). Surprisingly, we observed increased phago-cytosis of nuclear fragments even in patients with verylow uptake of immunoglobulin-opsonized beads (Figure2C). In the correlation analysis, there was no significantassociation between the phagocytosis of immunoglobulin-opsonized beads and nuclear fragments, for either gran-ulocytes or monocytes.
Role of anti-dsDNA autoantibodies in phagocy-tosis by blood-borne phagocytes of SNEC. We reconsti-tuted washed blood cells from normal healthy donorswith plasma from patients with SLE, which had beenshown to promote the uptake of SNEC. As shown inFigure 3A the high phagocytosis-supporting activity ofthe plasma from a patient with SLE was transferred tothe cells of the normal healthy donor. Furthermore, thecells of SLE patients were not rendered more capable of
Figure 2. Specific ingestion of SNEC by blood-borne phagocytes from patients with SLE. A and B,Phagocytosis indices (PhIx) of granulocytes and monocytes for immunoglobulin-opsonized fluo-rescent beads (A) and for propidium iodide–labeled SNEC (B), calculated as the product of thepercentage of fluorescent phagocytes and the mean fluorescence intensity of the phagocytes. C,Correlation of phagocytosis indices of immunoglobulin-opsonized beads and SNEC. Note that thegranulocytes and monocytes from some patients with SLE showed a significant reduction in theuptake of immunoglobulin-opsonized beads. However, significant uptake of SNEC by bothmonocytes and granulocytes was observed in two-thirds of these patients. In contrast, SNEC couldhardly be detected in monocytes or granulocytes from normal healthy donors. Phagocytosis ofSNEC was observed even in patients with a very low capacity for phagocytosis of immunoglobulin-opsonized beads. NS � not significant; RA � rheumatoid arthritis (see Figure 1 for otherdefinitions).
INFLAMMATORY CLEARANCE OF SECONDARILY NECROTIC CELLS IN SLE 1737
engulfing nuclear fragments when they were in thepresence of plasma from healthy donors (Figure 3A).Importantly, the levels of anti-dsDNA autoantibodiespositively correlated with the phagocytosis-supportingactivity of individual plasma samples (Spearman’s � �0.48, P � 0.001) (Figure 3B). We supplemented wholeblood from healthy donors with 2.5 mg/ml IgG fromhealthy donors or from SLE patients with phagocytosis-promoting activity. Healthy donor granulocytes incu-bated with the SLE-derived IgG fractions showed in-creased uptake of SNEC, while granulocytessupplemented with healthy donor–derived IgG showedno uptake of SNEC (Figure 3C).
Complement-dependent phagocytosis requiresthe action of Ca��-dependent proteases. To evaluatethe requirement for Ca�� for the engulfment by mono-cytes and granulocytes of SNEC, we analyzed wholeblood samples anticoagulated with EDTA and com-pared them with heparinized samples cultured under thesame conditions. The autoantibody-mediated uptake of
SNEC was completely inhibited in the presence ofEDTA (data not shown). To additionally verify theopsonization of SNEC by patient sera, we used indirectimmunofluorescence to test whether SNEC is recog-nized by human IgG. Figure 3D shows that SNEC wasopsonized exclusively by sera from patients with SLE.
Furthermore, phagocytosis by granulocytes ofSNEC opsonized with SLE anti-dsDNA autoantibodieswas strongly reduced when we used DNase I to degradethe DNA in the prey before adding it to whole bloodcultures (Figure 3E). Additionally, depletion of anti-dsDNA autoantibodies from a positive serum samplecompletely abolished the phagocytosis of SNEC bygranulocytes (Figure 3F).
Uptake of SNEC by isolated granulocytes andmonocytes in the presence of anti-dsDNA autoantibod-ies. We selected 6 serum samples from healthy donorsand 13 serum samples from patients with SLE (7 of 13were anti-dsDNA autoantibody positive) and used thesesera at 30% to supplement medium for the phagocytosis
Figure 3. Mediation of SNEC phagocytosis by anti–double-stranded DNA (anti-dsDNA) autoan-tibodies (AAb). A, Kinetics of phagocytosis by SNEC granulocytes from a representative normalhealthy donor and a patient with SLE. The phagocytosis-promoting activity was contained in theplasma of the patient, because cells from the patient did not take up SNEC in the presence ofplasma from a healthy donor. B, Significant bivariate nonparametric correlation between anti-dsDNA autoantibodies and phagocytosis indices for the uptake by granulocytes of SNEC. C,Phagocytosis in whole blood by SNEC granulocytes from a healthy donor before culture (brokenline) and after 4 hours of culture in the presence of 2.5 mg/ml IgG from a healthy donor (grey area)or a patient with SLE (solid line). D, Binding of SNEC to IgG from patients with SLE and controlsubjects. SNEC-binding autoantibodies were detected exclusively in patients with SLE. E, Reducedphagocytosis of SNEC opsonized with SLE anti-dsDNA autoantibodies by granulocytes when DNAin the prey was previously degraded. F, Abolished phagocytosis of SNEC opsonized with SLEanti-dsDNA autoantibodies by granulocytes by depletion of anti-dsDNA autoantibodies fromautoantibody-positive serum (SLE�). Bars in E and F show the mean and SD. PhIx � phagocytosisindex; PE � phycoerythrin (see Figure 1 for other definitions).
1738 MUNOZ ET AL
of SNEC by healthy donor phagocytes. Both granulo-cytes and monocytes were cocultured for 4 hours in thepresence of SNEC. Only sera positive for anti-dsDNAautoantibodies induced phagocytosis of SNEC by bothisolated granulocytes and monocytes (P � 0.01) (Figures4A and B, respectively). When we compared the phago-cytosis indices obtained with isolated cells with thosefrom the whole blood assays, they were highly signifi-cantly correlated for both cell types (P � 0.001) (Figures4C and D, respectively).
Role of autoantibody-mediated phagocytosis ofSNEC in secretion of inflammatory cytokines. We mea-sured cytokine secretion by blood-borne phagocytesinduced by the uptake of SNEC. Four hours after theaddition of SNEC, significantly elevated amounts of theinflammatory cytokines IL-8 (P � 0.025), IL-1� (P �0.025), TNF� (P � 0.025), IFN� (P � 0.037), and IL-18(P � 0.025) were produced (Figure 5A) by the patientswhose plasma contained phagocytosis-promoting anti-dsDNA autoantibodies, as determined by Farr assay(Figure 5D). Isolated granulocytes from healthy donorsproduced only IL-8 upon phagocytosis of SNEC, which
again was dependent on the presence of anti-dsDNAautoantibodies and SNEC (P � 0.004) (Figure 5B). Incultures of healthy donor–derived monocytes, the mostprominent cytokine was IL-1�, which was producedexclusively in the presence of anti-dsDNA autoantibod-ies and SNEC (P � 0.032) (Figures 5C and D).
DISCUSSION
We have established an ex vivo phagocytosisassay in whole blood where both cellular and humoralcomponents are present, which may participate in theclearance of particulate targets by blood-borne phago-cytes. The uptake of various particulate targets by gran-ulocytes and monocytes could, for the first time, bequantified in whole blood by flow cytometry. Moreover,by analyzing whole blood from patients at the momentof their medical visit, we are now able to get very closeto the in vivo picture of a patient’s actual clearancestatus.
Figure 4. Specific ingestion of secondarily necrotic cell–derived ma-terial (SNEC) by isolated blood-borne phagocytes from normalhealthy donors (NHDs) in the presence of anti–double-stranded DNA(anti-dsDNA) autoantibodies. A and B, Phagocytosis indices (PhIx) ofisolated granulocytes (A) and monocytes (B) for SNEC uptake,calculated as the product of the percentage of fluorescent positivephagocytes and the mean fluorescence intensity of the phagocytes.Isolated phagocytes from healthy donors were cultured for 4 hours at37°C in medium supplemented with 30% serum from 6 healthy donorsand 13 patients with systemic lupus erythematosus (SLE). SLE-derivedanti-dsDNA autoantibodies supported the phagocytosis of SNEC bygranulocytes and monocytes from healthy donors. C and D, Significantcorrelation between whole blood cultures and phagocytosis by isolatedcells of SNEC in the presence of sera from SLE patients.
Figure 5. Relationship between autoantibody-mediated phagocytosisof SNEC and secretion of inflammatory cytokines. A, Cytokine pro-duction in SLE patient–derived whole blood cultures in the presenceof SNEC (4 hours). Patients with high phagocytosis-promoting activityfor SNEC produced significantly higher amounts of the inflammatorycytokines interleukin-8 (IL-8), IL-1�, tumor necrosis factor � (TNF�),interferon-� (IFN�), and IL-18, but not the antiinflammatory cytokineIL-10. B, Production of IL-8 by isolated granulocytes from healthydonors upon phagocytosis of SNEC, in the presence of anti-dsDNAautoantibodies. C, Production of IL-1� by isolated monocytes fromhealthy donors after 18 hours of culture, in the presence of anti-dsDNA autoantibodies plus SNEC. D, Titers of anti-dsDNA asdetermined by Farr assay. NS � not significant (see Figure 4 for otherdefinitions).
INFLAMMATORY CLEARANCE OF SECONDARILY NECROTIC CELLS IN SLE 1739
We observed impaired phagocytosis ofimmunoglobulin-opsonized beads in some of the pa-tients with SLE. This is in agreement with our previousobservations made with monocyte-derived macrophages(15), in which clearance deficiencies in patients withSLE are discussed as being an important element for theinitiation of autoimmunity. The accumulation of cellcorpses and nuclear fragments observed in secondarylymphoid tissue (3) as well as in the bone marrow (16) ofsome patients with SLE may help to explain the loss ofboth peripheral and central B cell tolerance and theinduction of T cell help against self antigens.
In contrast to the uptake of immunoglobulin-opsonized beads, SNEC was taken up almost exclusivelyby granulocytes in at least 50% of the SLE patients. Theuptake of SNEC by blood-borne phagocytes has acertain similarity to the “LE cell phenomenon,” re-ported 60 years ago, exclusively in the bone marrow ofpatients with SLE. It was also revealed that phagocytesthat are present in bone marrow or buffy coats fromnormal healthy donors can be rendered to phagocytosenuclear material, by incubation with SLE plasma (17).More recently, the LE cell phenomenon was linked toantinuclear and antihistone H1 antibodies, respectively(18). LE cells (granulocytes that have taken up wholenuclei of dead cells) are seldom present in freshly drawnblood. This is most likely attributable to the fact thatblood-borne phagocytes are not easily able to engulfbig-sized prey such as whole nuclei. The small-sizedSNEC, however, is a nuclear “fast food” that is taken upby up to 80% of all phagocytes in the patient’s blood ina situation that is close to the in vivo situation.
Surprisingly, we observed increased phagocytosisof nuclear fragments even in patients with very lowuptake of immunoglobulin-opsonized beads. In the cor-relation analysis, there was no significant associationbetween the phagocytosis of immunoglobulin-opsonizedbeads and nuclear fragments, for either granulocytes ormonocytes. This suggests that even the low phagocytoticcapacity displayed by some SLE patients forimmunoglobulin-opsonized beads (and certain otherparticles such as albumin-coated beads, zymosan, bacte-ria, and acetylated LDL) (data not shown) is sufficientfor the uptake of SNEC—a prey that is ignored byphagocytes from control subjects.
A plethora of inflammatory manifestations arepresent in patients with SLE. Such symptoms are oftenmediated by autoantibodies. Such autoantibodies aremainly directed against nuclear components, which aremodified or translocated in dead and dying cells (1).DNA-containing nucleosomes are released in high
amounts into the circulation of patients with SLE,leading to immune complex formation with anti-dsDNAautoantibodies of the IgG isotype (8). In healthy donors,the clearance of apoptotic material is very rapid, effi-cient, and antiinflammatory (2), preceding the release ofnuclear constituents from apoptosing cells. In the case ofSLE patients, the low phagocytic activity as well as thepresence of nuclear autoantigens, autoantibodies, andabnormally low levels of complement may drive theclearance process toward Fc receptor (FcR)–mediatedinflammation and tissue damage (9,19,20).
This study is the first to demonstrate that anti-dsDNA autoantibodies from patients with SLE promotephagocytosis by blood-borne phagocytes of SNEC. IgGbinding to SNEC correlates with anti-dsDNA autoanti-bodies. Extractable nuclear antigen (ENA) reactivitywas also included in the analysis of our data. However,ENA reactivity alone was not associated with promotionof phagocytosis. Some of the anti-dsDNA antibody–positive patients also had ENA reactivity and were notdistinguishable in the phagocytosis-promoting activityfrom those with only anti-dsDNA reactivity (data notshown). Autoantibodies such as those promoting apo-ptotic and necrotic cell phagocytosis have been reportedby our group and by other investigators (21–24). Theseobservations were made in vitro for monocyte-derivedmacrophages and apoptotic and necrotic tumor cell lines.
In our experiments, the chelator of divalent cat-ions, EDTA, inhibited the IgG-mediated phagocytosis ofSNEC by both monocytes and granulocytes. EDTA issupposed to work by inhibiting activation of the classicalpathway of complement by SNEC immune complexes,which is dependent on Ca�� and Mg��. Therefore, weconclude that opsonization with IgG autoantibodies andcomplement is required for the uptake of SNEC byblood-borne phagocytes. Similarly, autoantibodies suchas anti–�2-glycoprotein I are capable to poise activatedplatelets in a phosphatidylserine-exposing status,thereby favoring their phagocytic clearance (25). In theabsence of autoantibodies, phagocytosis of late apopto-tic cells is antibody independent and mediated solely bycomplement and other adaptor molecules through inte-grin receptors (26). In contrast to FcR-mediated phago-cytosis, this does not elicit inflammatory responses (27).
We corroborated our observations in wholeblood when we cultured isolated normal phagocytes inthe presence of sera from patients with SLE. Further-more, we used an affinity-purified monoclonal auto-antibody from a patient with SLE (antibody 33.C9) insome experiments (28). We observed a dose-dependentpromotion of the uptake of SNEC by granulocytes and
1740 MUNOZ ET AL
monocytes when adding 33.C9 to the whole blood ofhealthy donors (data not shown). As we previouslyshowed, SNEC binds and activates complement inde-pendent of autoantibodies (29) and also binds furtherdying and dead cell–related ligands (annexin V, CRP,acetylated LDL, and N pseudonarcissus lectin).
Serum DNase I also plays an important role inthe processing of nuclear fragments released from dyingcells (14). The above-mentioned molecules, and proba-bly others, orchestrate the rapid and efficient removal ofdying cells from tissues without eliciting inflammation.Alterations in �1 of these molecules have been pro-posed as being responsible for the clearance deficiencyin patients with SLE (30). However, circulating phago-cytes ignore SNEC in the absence of opsonizing IgG. Incontrast, in SLE patients with circulating autoantibodies,SNEC is directed into blood-borne phagocytes, mostlikely by anti-dsDNA autoantibodies. This may haveserious implications for the perpetuation of inflamma-tory responses in these patients.
Autoantibody-mediated phagocytosis of SNECinduced secretion of the inflammatory cytokines IL-8,IL-1�, TNF�, IFN�, and IL-18 by whole blood culturesof the patients. This complex cytokine profile may resultfrom the interplay with many other cell types present inthe system (e.g., plasmacytoid dendritic cells, B cells andT cells, natural killer cells). Experiments performed byHeyder et al (31) revealed that plasmacytoid dendriticcells are the main producers of INF� when exposed toapoptotic bodies or SNEC. After autoantibody-mediated phagocytosis of SNEC, isolated granulocytesproduced solely IL-8, and isolated monocytes producedsolely IL-1�. Therefore, we conclude that nucleic acidsare important triggers of the cytokine responses oncethey reach intracellular receptors shuttled by autoanti-bodies. To our knowledge, this is the first detaileddescription of anti-dsDNA autoantibody–induced in-flammation mediated by blood-borne phagocytes exvivo. We propose a role of SNEC–anti-dsDNA immunecomplexes in the perpetuation of inflammation in thecirculation of patients with SLE, contributing to thepathogenesis of this disease.
In the in vivo situation, autoantibodies may rec-ognize an excess of nuclear material in the circulationand thereby contribute to an inflammatory disposal ofcellular debris in vessels. Cells from the innate immunesystem are a major source of inflammatory cytokines.They modulate the course of the immune response afterencountering foreign or altered self antigens. Activatedphagocytes producing inflammatory cytokines have alsobeen detected in the irradiated skin of patients with
SLE, but an association with autoantibodies was notreported (32). Monoclonal anti-dsDNA antibodies aloneare described to induce production of inflammatorycytokines in human endothelial cells, neutrophils, andPBMCs in vitro (33–35). Anti-dsDNA immune com-plexes are also able to induce specifically the productionof IFN� in PBMCs (6) and U1 small nuclear RNPimmune complexes in plasmacytoid dendritic cells. Thisis the first study to show that anti-dsDNA autoantibod-ies massively promote phagocytosis of SNEC and subse-quently produce high amounts of inflammatory cyto-kines that may thus drive the persistent and recurrentinflammatory manifestations of SLE.
Overloading cells with SNEC that normally arenot accustomed to handling this kind of prey may haveserious implications for the pathogenesis of SLE. Asrecently observed in DNase II–knockout mice, undi-gested DNA in lysosomes may leak into the cytoplasmand cause the production of type I IFN (36). If anoverload of SNEC results in leakage of DNA into thecytoplasm, this may induce IFN� and contribute to thetype I IFN signature frequently observed in the PBMCsof patients with SLE (37). Interestingly, we observedIFN� in the supernatants of whole blood cells, suggest-ing that circulating plasmacytoid dendritic cells alsorespond to SNEC in the presence of anti-dsDNA auto-antibodies. These cells have been shown to be the majorproducers of type I IFN in blood (6).
In conclusion, clearance deficiencies may be re-sponsible for the loss of tolerance and the initiation ofautoimmunity to nuclear autoantigens in patients withSLE. In established disease, increased autoantibody-dependent phagocytosis of cellular remnants may initi-ate an amplification loop of inflammation. This contrib-utes to the chronicity of the autoimmune response,providing further stimuli and survival signals for autore-active T cells and B cells.
AUTHOR CONTRIBUTIONSAll authors were involved in drafting the article or revising it
critically for important intellectual content, and all authors approvedthe final version to be published. Dr. Gaipl had full access to all of thedata in the study and takes responsibility for the integrity of the dataand the accuracy of the data analysis.Study conception and design. Munoz, Frey, Kern, Kalden, Herrmann,Gaipl.Acquisition of data. Munoz, Janko, Grossmayer, Herrmann, Gaipl.Analysis and interpretation of data. Munoz, Janko, Grossmayer, Frey,Voll, Schett, Fietkau, Herrmann, Gaipl.
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