Download Lack of Chromatin and Nuclear Fragmentation In Vivo Impairs the

Lack of Chromatin and Nuclear
Fragmentation In Vivo Impairs the
Production of Lupus Anti-Nuclear Antibodies
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of August 2, 2017.
Lorenza Frisoni, Lenese McPhie, Sun-Ah Kang, Marc
Monestier, Michael Madaio, Minoru Satoh and Roberto
Caricchio
J Immunol 2007; 179:7959-7966; ;
doi: 10.4049/jimmunol.179.11.7959
http://www.jimmunol.org/content/179/11/7959
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References
The Journal of Immunology
Lack of Chromatin and Nuclear Fragmentation In Vivo
Impairs the Production of Lupus Anti-Nuclear Antibodies1
Lorenza Frisoni,* Lenese McPhie,* Sun-Ah Kang,† Marc Monestier,† Michael Madaio,†
Minoru Satoh,‡ and Roberto Caricchio2*
D
ying cells have been proposed as a critical reservoir of autoantigens in systemic lupus erythematosus (SLE)3 (1). Indeed, impaired clearance or excessive production of apoptotic cells or their debris can lead to SLE-like disease in certain
mouse models (2–7). Moreover, lupus patients exhibit increased circulating apoptotic debris that correlates with disease activity (8).
Both necrotic and apoptotic cells release chromatin (9, 10). In
particular, an important mechanism by which apoptotic cells are
thought to produce nuclear autoantigens in SLE is through chromatin fragmentation (11), which allows subsequent fragmentation
of the nucleus (12). Fragmented nuclei and their contents concentrate into characteristic blebs and bodies, which are extrusions of
the cytoplasmic membrane and are of suitable size to be taken up
and cross-presented by immature dendritic cells (1, 13). Finally,
chromatin fragmentation allows apoptotic cells to release nuclear
material such as fragmented chromatin itself and RNA/protein
*Division of Rheumatology, Department of Medicine, University of Pennsylvania,
and †Department of Microbiology and Immunology, and Division of Nephrology,
Temple University School of Medicine, Philadelphia, PA 19140; and ‡Division of
Rheumatology and Clinical Immunology, Department of Medicine, and Department
of Pathology, Immunology, and Laboratory Medicine, University of Florida, Gainesville, FL 32610
Received for publication August 1, 2007. Accepted for publication September
19, 2007.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by the Lupus Research Institute (R.C.), the National Institutes of Health/National Institute of Arthritis and Musculoskeletal and Skin Diseases (R.C), and the American Heart Association (M.M).
2
Address correspondence and reprint requests to Dr. Roberto Caricchio, Division of
Rheumatology, University of Pennsylvania, 751 Biomedical Research Building II/III, 421
Curie Boulevard, Philadelphia, PA 19104. E-mail address: [email protected]
3
Abbreviations used in this paper: SLE, systemic lupus erythematosus; aCL, anticardiolipin; CAD, caspase-activated DNase; EAE, experimental autoimmune encephalomyelitis; NP40, Nonidet P-40; ANA, anti-nuclear Abs; snRNP, small nuclear ribonucleoproteins; MRL/lpr, MRL/Mp-lpr/lpr; IP, immunoprecipitation technique;
pristane, 2,6,10,14-tetramethylpentadecane; RT, room temperature; CL,
chemiluminescence.
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
www.jimmunol.org
complexes into the circulation, two major lupus autoantigens,
which provide the autoantigens that sustain an ongoing autoimmune response (14 –16).
Many studies have supported, directly or indirectly, the relevance of apoptotic molecular steps, such as chromatin fragmentation, in lupus autoimmunity; however no direct in vivo evidence
has yet been presented to support such a hypothesis. The apparent
difficulty of creating a fragmented-chromatin free immune system
has seriously limited studies in such direction. The recent development of a new mutant mouse deficient for caspase-activated
DNase (CAD), also known as DNA fragmentation factor 40 (17),
has offered the opportunity to study the source of nuclear autoantigens (18) in vivo. Indeed, this mouse displays an impaired ability
to fragment chromatin at the internucleosomal level, to fragment
the nucleus, and to induce apoptotic membrane blebbing and body
forming (for a complete review see Refs. 19, 20). Nevertheless,
important upstream apoptotic steps, such as exposure of membrane
phospholipids, are preserved (21).
Therefore, we chose this mouse model to study, directly, the in
vivo role of apoptotic molecular steps in promoting or sustaining
SLE autoimmunity. We used, for our studies, an inducible model
of lupus triggered by a single injection of pristane (2,6,10,14-tetramethylpentadecane) oil (22, 23). Pristane-induced lupus is characterized by many typical lupus features, including production of
anti-chromatin Abs and other anti-nuclear Abs (ANA), polyclonal
B cell activation, and mild to severe glomerulonephritis, depending on the mouse strain (22, 23). Moreover, apoptotic cell death is
an important mechanism in the pristane-induced lupus model (24).
Indeed, we found that in this lupus model, in the absence of
apoptotic fragmented chromatin, production of anti-chromatin, antismall nuclear ribonucleoproteins (snRNPs), and other nucleus-directed autoantibodies was severely impaired or completely absent,
while production of Abs directed at membrane lupus autoantigens,
such as cardiolipin (25), was preserved. Finally, in this lupus
model, the absence of chromatin fragmentation did not protect the
mice from developing kidney immune-complexes deposition and
mild lupus nephritis.
Downloaded from http://www.jimmunol.org/ by guest on August 2, 2017
Nuclear autoantigens in systemic lupus erythematosus are thought to derive primarily from apoptotic cells, yet there is no direct
evidence that interfering with apoptosis impairs the generation of lupus autoantibodies. Here we use a mouse model that lacks the
endonuclease caspase-activated DNase (CAD), resulting in an absence of chromatin and nuclear fragmentation during apoptotic
cell death. We show that in this mouse, production and release into circulation of chromatin is impaired after exposure to several
apoptotic triggers, but that the absence of CAD does not interfere with upstream steps of apoptosis or immune system function.
Finally we show that in CAD-mutant mice, impaired lupus autoimmunity is skewed toward known cytoplasmic components, and
autoimmunity toward membrane autoantigens is preserved, while autoimmunity toward chromatin and other lupus nuclear
targets is severely impaired or absent. We also show, as control, that the induction of experimental autoimmune encephalomyelitis
is not affected by the absence of CAD. Thus, our work in vivo strongly suggests that apoptotic molecular steps during cell death
generate nuclear autoantigens to sustain the specific autoimmune response in systemic lupus erythematosus. The Journal of
Immunology, 2007, 179: 7959 –7966.
7960
LACK OF CHROMATIN FRAGMENTATION IMPAIRS LUPUS ANA
Thus, our work in vivo strongly suggests that apoptotic molecular steps during cell death generate nuclear autoantigens to sustain the specific autoimmune response in SLE.
Materials and Methods
Mice
CAD⫺/⫺ mice were generated on the 129 background as previously described (18) and were a generous gift of Dr. Nagata (Osaka University,
Japan). They were then backcrossed at least eight generations with
C57BL/6 mice to eliminate the 129 background. Mice were bred and maintained in accordance with the guidelines of the University Laboratory Animal Resource Office of the University of Pennsylvania, an American Association for the Accreditation of Laboratory Animal Care accredited
facility.
Induction of lupus-like autoimmunity
CAD⫹/⫹ and CAD⫺/⫺ mice were injected i.p. with 0.5 ml of pristane to
induce a lupus-like syndrome (22). Serum samples were collected before
the injection, at 2 wk, and then monthly until the mice were sacrificed.
Induction of cell death
DNA isolation
Plasma samples were obtained by collecting blood in a microcentrifuge
tube containing 50 IU of heparin followed by spinning at 2000 rpm for 5
min. The pellet was saved and the plasma was filtered through a 0.2 ␮ filter
to eliminate residual cells. DNA was isolated from cells pellet, supernatant,
or plasma using the Qiagen DNA isolation kit (Qiagen) and was analyzed
by electrophoresis on a 2% agarose gel containing 0.5 ␮l/ml ethidium
bromide.
ELISA
Anti-OVA ELISA
Mice were immunized IP with 100 ␮g per mouse of OVA in PBS emulsified with CFA. To assess the titer of specific anti-OVA Abs produced,
microtiter plates were coated O/N at 4°C with 100 ␮l of 10 ␮g/ml OVA
(Sigma-Aldrich) solution in 0.05 M carbonate buffer. All washes were
performed in PBS/0.05% Tween 20. To avoid nonspecific binding, plates
were incubated in 3% BSA in PBS/0.05% Tween 20 solution for 1 h at RT.
Sera were diluted 1:2000 in PBS/Tween and 50 ␮l were incubated for 90
min at RT. Dilutions of a monoclonal anti-OVA Ab (Sigma-Aldrich) were
used to generate a standard curve. After washing, a secondary F(ab⬘)2 goat
anti-mouse IgG (Fc␥), alkaline phosphatase conjugated (Jackson ImmunoResearch Laboratories) was used at 1:10000 dilution in PBS/Tween20.
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In vivo, systemic apoptosis was induced by exposure to 600 or 1500 Rads
using a Cs-137 emission source of ␥ radiation. Mice were then sacrificed
at 2.5 or 8 h after radiation exposure according to the experimental conditions. Organs (thymus, spleen, bone marrow, and liver) were explanted
and processed for DNA extraction.
Alternatively, apoptosis was induced in the form of septic shock caused
by the i.p. injection of LPS (5 ␮g per mouse) and D-Galactosimine (20 mg
per mouse). Mice were then sacrificed at 6 h post injection.
To induce organ specific apoptosis, anti-Fas Ab was injected in the
hepatic portal vein. Fully anesthetized mice underwent surgery to expose
the abdominal cavity and the portal vein. Anti-Fas Ab (Jo2, 5 ␮g per
mouse; BD Pharmingen) was delivered slowly in 200 ␮l of sodium chloride solution (0.9%) with a 28G insulin syringe directly into the portal vein.
Mice were sacrificed 5 h after injection and the liver explanted, fixed in
formalin, and processed for H&E staining (26).
In vitro, apoptosis was induced on cell cultures of splenocytes by addition of Staurosporine or Cycloheximide at the concentration of 100 ␮M
and 30 ␮g/ml, respectively. Apoptosis was also induced by overnight starvation (serum free medium) and UV B irradiation (20 mJ/cm2). Splenocytes were seeded at 1.5 ⫻ 106 cells per well in a six well dish in a total
volume of 1 ml. At 2, 6, and 24 h after treatment, cells were separated from
the supernatant by low speed centrifugation (⬍2000rpm) and supernatant
was further filtered to eliminate contaminating cells as described above.
Membrane flipping and phospholipids exposure was assessed by annexin
V-PE staining according to the manufacturer’s instruction (BD Biosciences). In brief, cells were washed twice with PBS and resuspended in
100 ␮l of annexin V buffer. Five ␮l of annexin V was added and incubated
in the dark for 15 min at room temperature (RT). Samples were read within
1 h. In some experiments, flow cytometric scatter (forward light scatter vs
side light scatter) was used as a measure of apoptotic cell death and debris
generation (27, 28).
Anti-chromatin and total Ig ELISA
Microtiter plates were coated with 100 ␮l of 1 ␮g/ml chicken chromatin in
borate-buffered saline (200 mM boric acid and 75 mM NaCl, pH 8.4) at
4°C for 16 h. Wells were washed with NET/Nonidet P-40 (NET; 0.15 M
NaCl, 2 mM EDTA, and 50 mM Tris-HCl, pH 7.5, 0.3% NP40) and
blocked with 0.5% BSA in NET/Nonidet P-40 for 1 h at 22°C. Wells were
then incubated with 100 ␮l of 1:500 mouse sera in same buffer for 1 h at
22°C, washed three times with NET/Nonidet P-40, and incubated with 100
␮l alkaline phosphatase-conjugated goat anti-mouse IgG, Fc␥ fragment
(Jackson ImmunoResearch Laboratories, dilution 1:1000) in blocking
buffer for 1.5 h at 22°C. After washing, the plates were developed with
p-nitrophenyl phosphate substrate (Sigma-Aldrich). OD405 was converted
to units based on a standard curve produced by serial dilutions of pooled
sera from MRL/Mp-lpr/lpr (MRL/lpr) mice: 1/500 dilution ⫽ 625 U;
1/2500 ⫽ 125 U; 1/12500 ⫽ 25 U; 1/62500 ⫽ 5 U; 1/312500 1 U; and
1/1562500 0.2 U. Usually, the standard is clearly positive at a 1/312500
dilution. For the ELISA to measure total Ig levels, a similar protocol was
used: plates were coated with three ␮g/ml goat anti-mouse k/␭ L chain Abs
in ratio 9:1, sera were diluted 1:200,000, and as secondary Ab a 1:1000
dilution of alkaline phosphatase-labeled goat anti-mouse Abs specific for
IgG1, IgG2a, IgG2b, IgG3, IgA, and IgM were used. Standard curves were
generated by dilution of commercially available Ig isotype standards from
100 ng/ml to 0.5 ng/ml.
Anti-cardiolipin ELISA
Anti-Cardiolipin (CL) serum levels were detected according to a previously described protocol (25). In brief, polystyrene microtiter plates (BD
Biosciences) were coated with 20 ␮g/ml CL in ethanol. After blocking,
sera were diluted 1/100 in PBS containing 1% BSA and 0.05% Tween 20
and incubated for 2 h at RT. Serial dilutions of the purified anti-CL mAb
FB1 (mouse IgG2b) obtained from a 5-mo-old female (New Zealand
White ⫻ BXSB) F1 mouse were used as a standard curve (25). After
washing, binding was detected with goat anti-mouse IgG-alkaline phosphatase (Southern Biotechnology Associates) followed by color development with the appropriate substrate.
Immunofluorescent ANA
Staining was performed on prefixed HEp-2 cells on glass slides following
manufacturer instruction (Antibodies Incorporated). In brief, mouse sera
were used at 1/40 and 1/160 dilution in PBS and incubated for 30 min at
RT in a humidified chamber. After washing, ANA were detected with a
goat anti-mouse IgG (Fc␥ specific) FITC-conjugated Ab, incubated for 30
min in the dark. Slides were washed again, dried, and mounted with
Vectashield (Vector Laboratories). Images were taken with a Nikon TE300
scanning confocal microscope, equipped with Nomarski differential interphase contrast optics, coupled to the Bio-Rad Radiance 2000 laser. A ⫻60
magnification was used.
Immunoprecipitation
Autoantibodies to cellular proteins in murine sera were analyzed by immunoprecipitation of [35S]methionine/cysteine-radiolabeled K562 cell extract using 5 ␮l of murine serum and SDS-PAGE (22, 23). In brief, cells
were labeled with [35S]methionine/cysteine (93 ␮Ci/ml), lysed in 0.5 M
NaCl NET/NP40 buffer containing 0.5 mM PMSF and 0.3 trypsin inhibitory units/ml aprotinin. After centrifugation, the cleared extract was immunoprecipitated with protein A-Sepharose beads coated with 5 ␮l of
mouse sera. Immunoprecipitates were washed three times with 0.5 M NaCl
NET/NP40, washed once with NET buffer, and finally analyzed with SDSPAGE (12.5% SDS) and autoradiographed. Abs specificity was assessed
using reference sera.
Flow cytometry
Single cell suspension was washed in cold PBS and Fc␥R was blocked
with 2.4G2 Ab for 10 min on ice. The following mAbs purchased from BD
Pharmingen were used for staining: Rat IgG2a PerCP or allophycocyanin
-conjugated anti-CD19, Rat IgG2a biotin-conjugated anti-B220, Armenian
hamster IgG1 R-PE-conjugated anti-CD3, Rat IgG2a PE anti-CD4, Rat
IgG2a FITC-conjugated anti-CD8a (Ly-2), Rat IgG2a biotin-conjugated
anti-CD11b, Rat IgG2b FITC anti-CD21, Rat IgG2a PE anti-CD23, and
Armenian Hamster IgG1 allophycocyanin anti-CD11c. Cells were incubated with directly labeled Abs for 30 min and washed. An additional 20
min incubation with streptavidin-allophycocyanin or PerCP was performed
to detect biotinylated Abs. After staining, cells were washed in PBS, fixed
in 1% paraformaldehyde in PBS and analyzed on a BD Bioscience FACSCalibur. For active Caspase-3 staining (intracellular staining), cells were
permeabilized using Cytofix/Cytoperm kit (BD Biosciences) for 20 min on
ice and the pellet was washed twice in intercellular staining buffer (IBS:
PBS, 0.1% azide, 3% FCS, and 0.1% saponin). Cells were then stained
with rabbit anti-caspase-3 mAb (BD Biosciences) diluted in IBS buffer for
30 min at RT at the concentration of 0.25 ␮g/1⫻106 cells. After further
The Journal of Immunology
7961
Results
Apoptotic chromatin and nuclear fragmentation, and formation
of apoptotic bodies are absent in CAD⫺/⫺ mice
Release of nuclear contents during apoptotic cell death has been
suggested as a fundamental step to provide autoantigens in SLE
(2). To demonstrate the validity of the CAD⫺/⫺ mouse as a model
in which nuclear contents are not released, we induced apoptosis in
vitro in splenocytes from CAD⫹/⫹ and CAD⫺/⫺ mice. Intracellular chromatin fragmentation and its extracellular release were
evaluated over time by the presence of DNA laddering, a direct
measure of fragmented chromatin (11) (Fig. 1A). Results in Fig. 1A
show that, in contrast to CAD⫹/⫹ splenocytes, no DNA laddering
FIGURE 1. CAD⫺/⫺ mice do not exhibit fragmented DNA either in
vivo or in vitro upon apoptosis induction. CAD⫹/⫹ and CAD⫺/⫺ splenocytes were cultured in six well dishes (2⫻106 per well in 2 ml of complete
RPMI medium) in the presence of cycloheximide and staurosporine to
induce apoptosis. At different time points, cells were collected by centrifugation and the supernatant was filtered through a 0.2 ␮m syringe filter to
remove residual cells. DNA was extracted from both cell pellet and supernatant, and run on a 2% agarose gel. A, DNA fragmentation was visible
already at 2 h after the beginning of the treatment in the cell extracts from
CAD⫹/⫹ mice and at 24 h in supernatant extracts. No visible DNA fragmentation was detected in either cell or supernatant extracts from
CAD⫺/⫺ mice. B, Anti-Fas Ab was injected directly into the portal vein
to induce organ specific apoptosis. Five hours after anti-Fas treatment,
mice were sacrificed and the liver was fixed in 10% buffered formalin in
PBS and processed for H&E staining. CAD⫹/⫹ mice showed hepatocytes
with fragmented nuclei, while CAD⫺/⫺ mice showed condensed nuclei
(magnified inset). C, Splenocytes isolated from CAD⫹/⫹ and CAD⫺/⫺
mice were treated to induce cell death and incubated overnight (serum-free
medium for starvation; 20 mJ/cm2 for UV B irradiation). In CAD⫹/⫹
mice, a large population of cells with low forward and side scatter (forward
light scatter and side light scatter) indicates that the cells underwent fragmentation into smaller blebs and bodies (p ⬍0.05 for both treatments). This
population is less represented in CAD⫺/⫺ mice, showing that cellular
breakup does not occur in vitro. D, Mice received 600 rads of ␥ radiation
to induce systemic apoptosis in vivo. Thymocytes and splenocytes from
CAD⫹/⫹ and CAD ⫺/⫺ showed a similar percentage of annexin V positive cells, both constitutively (no radiation) and upon induction of apoptosis (with radiation) (left panel). Activation of Caspase-3 at 4.5 h after
irradiation was also comparable (right panel). E, CAD⫺/⫺ and ⫹/⫹ littermates were treated with either ␥-irradiation or LPS/D-Galactosimine.
CAD⫹/⫹ exhibited fragmented DNA in all the tissues tested (thymus, T;
spleen, S; bone marrow, BM; liver, L) already, 2.5 h after treatment. In
contrast, no DNA fragmentation was visible in the organs explanted from
CAD⫺/⫺ mice. Untreated mice were used as control. F, Plasma DNA was
collected from 10 CAD⫹/⫹ and 10 CAD⫺/⫺ mice 8 h after ␥-irradiation.
Plasma was pooled, filtered to eliminate residual cells, and DNA was extracted. DNA fragments were released in the circulation of CAD⫹/⫹ mice
but were not detectable in CAD⫺/⫺ mice.
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washing, cells were stained with a secondary Ab, goat anti-rabbit FITCconjugated (Jackson ImmunoResearch Laboratories) at 1:100 dilution in
IBS. Cells were fixed in 1% paraformaldehyde and analyzed by flow
cytometry.
Experimental autoimmune encephalomyelitis (EAE) induction
Two-month-old CAD⫹/⫹ and CAD⫺/⫺ mice were immunized subcutaneously with 0.2 ml of myelin oligodendrocyte glycoprotein peptide (200
␮g in PBS) and CFA (200 ␮g) emulsion. After anesthesia, mice were
injected in the four areas close to the axillary and inguinal lymph nodes.
Pertussis toxin (200 ng in 200 ␮l of PBS) was also administered i.p. at the
time of immunization and 48 h later. Neurological signs were monitored
for up to 3 wk and scored according to the guidelines in Current Protocol
of Neuroscience (29). In brief, mice were monitored and scored for the
following abnormalities: no abnormalities (score 0), a limp tail (score 1),
mild hind limb weakness (score 2), severe hind limb weakness (score 3),
complete hind limb paralysis (score 4), quadriplegia or moribund state
(score 5), and death (score 6) (29).
Detection of immune deposits
Whole kidneys were frozen in OTC medium and cut in 4 ␮m thick slices.
Slices were air dried and washed in PBS 3⫻ for 5 min. Fixation was
performed with acetone at ⫺20°C for 20 min. After washing in PBS, specimens were incubated with goat anti-mouse IgG (Fc␥ specific) FITC-conjugated Ab diluted 1/75 in PBS. Slides were washed again, dried, and
mounted with Vectashield (Vector Lab) (30).
Grading the severity of nephritis
H&E staining was performed and a previously described method was used
to assess the disease severity. A grade higher than 2⫹ is considered disease
(31).
Statistical analysis
Mann-Whitney or Wilcoxon matched pair test and ␹2 test were used to
determine the statistical significance of differences in values and frequency
between groups, respectively. Analyses were conducted with GraphPad
Prism 4.0c software for Mac (GraphPad).
7962
LACK OF CHROMATIN FRAGMENTATION IMPAIRS LUPUS ANA
Table I. Lymphocyte subpopulation in CAD⫺/⫺ mice
CAD⫹/⫹
CD19
B220
CD3
CD4
CD8
CD11b
CD19/CD21⫹
CD19/CD23⫹
CD11b/CD11c⫹
CD19/MHCII⫹
CD19/CD80⫹
CD19/CD86⫹
CD4/CD69⫹
CD8/CD69⫹
a
CAD⫺/⫺
46.4 ⫾ 1.5
57.4 ⫾ 5.0
26.0 ⫾ 5.3
17.3 ⫾ 0.8
11.3 ⫾ 2.5
9.4 ⫾ 3.5
10.4 ⫾ 1.0
66.6 ⫾ 8.2
0.8 ⫾ 0.2
60.6 ⫾ 19.1
6.3 ⫾ 0.9
29.9 ⫾ 1.4
72 ⫾ 17.7
85 ⫾ 2.2
a
44.0 ⫾ 5.9
53.7 ⫾ 1.0
33.9 ⫾ 3.2
18.1 ⫾ 3.7
13.6 ⫾ 1.2
7.12 ⫾ 0.3
12.3 ⫾ 3.5
67.7 ⫾ 3.6
1.0 ⫾ 0.1
55.2 ⫾ 10.3
8.1 ⫾ 2
25.7 ⫾ 4.5
64.7 ⫾ 32.3
80.8 ⫾ 8.7
Indicates percentage of positive cells ⫾ SD.
CAD⫺/⫺ mice have a functional immune system
Kawane et al. have previously reported that CAD null mice, as
opposed to DNase II null mice, have normal thymic development
(18). Nevertheless, the immune system in the absence of chromatin
fragmentation has been only partially investigated (18). We therefore first determined whether CAD⫺/⫺ mice differed from their
CAD⫹/⫹ littermates in the general composition of immune competent cells, and then if the absence of CAD interfered with a T
cell-dependent humoral immune response. No statistically significant differences in percentage were seen among numbers of
splenic T cells, B cells, and macrophages from CAD⫹/⫹ and
CAD⫺/⫺ mice (Table I). Likewise, no differences emerged when
more specific populations were targeted, i.e., follicular (CD23/
CD19 positive) and marginal zone (CD21/CD19 positive) B cells
(Table I). B, T, and dendritic cell activation parameters before or
after stimulation in vivo were also equally represented (Fig. 2A,
Table I).
Next, we tested the capacity of CAD⫺/⫺ mice to mount an
immune response against a foreign Ag. We immunized CAD⫹/⫹
and CAD⫺/⫺ mice with OVA in CFA and evaluated the production of anti-OVA Abs by specific ELISA at 2, 3, and 4 wk after
immunization. Both CAD⫹/⫹ and CAD⫺/⫺ mice were able to
build a vigorous immune response as indicated by the high IgG
anti-OVA Ab levels as early as 2 wk after immunization (Fig. 2B).
We also tested the ability of CAD⫺/⫺ mice to develop EAE, a
model of autoimmune disease that does not depend on apoptotic
cells as a source of Ag (38). We found no statistical differences in
the EAE clinical score between the CAD⫹/⫹ and the CAD⫺/⫺
mice (Fig. 3). Although abnormalities are still possible, based on
our results, we concluded that the absence of chromatin fragmentation does not grossly interfere with the immune functions of a
normal immune system and that the absence of CAD does not
prevent development of EAE.
FIGURE 2. CAD⫺/⫺ mice have a functional immune system.
CAD⫺/⫺ and CAD⫹/⫹ mice were injected with LPS to induce systemic
apoptosis. We investigated splenic B cell in vivo activation 24 h later by
staining for activation markers. A, The statistical analysis shows no difference in up-regulation of the activation marker CD69 between the
CAD⫺/⫺ and CAD ⫹/⫹ CD19 positive subsets. B, Five CAD ⫹/⫹ and
5 CAD⫺/⫺ mice were immunized with 100 ␮g of OVA in PBS emulsified
with an equal volume of CFA. Mice were bled at the times indicated and
sera were tested by ELISA for anti-OVA Ab levels. CAD⫺/⫺ mice responded to the immunogenic challenge as well as CAD⫹/⫹ mice, as
shown by the comparable levels of Abs produced (p ⫽ n.s.).
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was detectable in the CAD⫺/⫺ splenocytes at any time-point, indicating that intracellular chromatin fragmentation is impaired in
this mouse. Moreover, apoptotic chromatin was detected only in
the supernatant from CAD⫹/⫹ splenocytes, while in the
CAD⫺/⫺ splenocytes there was no detectable DNA laddering,
even 24 h after the induction of cell death.
Apoptotic bodies concentrate nuclear content, expose autoantigens, and are of the appropriate size for uptake by professional
APCs (13). For these reasons, apoptotic bodies are an important
source of autoantigens in lupus. We therefore evaluated whether
production of apoptotic bodies was impaired in CAD⫺/⫺ cells.
Fig. 1B shows liver sections after in vivo induction of cell death
with anti-Fas (32). Hepatocytes with fragmented nuclei were
present only in CAD⫹/⫹ mice, while only condensed nuclei were
found in CAD⫺/⫺ mice. Fig. 1C shows, using flow cytometry,
apoptotic changes in splenocytes after stimuli, such as starvation or
UV B irradiation. In particular, the formation of apoptotic bodies
is demonstrated by the reduction of size and density in the forward
side scatter plots (33). As previously shown by others, only
CAD⫹/⫹ cells formed apoptotic bodies, while CAD⫺/⫺ did not
undergo classic apoptotic morphological changes. To demonstrate
that, in CAD⫺/⫺ mice, other fundamental apoptotic features are
retained even when DNA fragmentation is impaired, cell membrane flipping and phospholipids residue exposure, and caspase-3
activation on both CAD⫺/⫺ and CAD⫹/⫹ apoptotic splenocytes
were determined (Fig. 1D). Apoptotic splenocytes from both
CAD⫹/⫹ and CAD⫺/⫺ mice showed an equal capacity of binding for annexin V (Fig. 1D, left panel), an important marker of
membrane flipping during apoptotic cell death (34). These results,
along with previous published data (35), suggest that the impairment of DNA fragmentation does not affect the triggering of
phagocytosis by professional scavengers, one of the basic features
of apoptosis (35, 36). Apoptotic splenocytes in both mice also
showed similar caspase-3 activation (Fig. 1D, right panel), demonstrating comparable activation of this fundamental apoptotic
molecular step (37).
We also evaluated chromatin fragmentation in vivo using gamma-irradiation and D-galactosamine/LPS to induce apoptosis.
DNA laddering was used to assess intracellular fragmentation of
apoptotic chromatin in the tissues and the circulation. Several organs were harvested before and after the apoptotic stimuli. As
shown in Fig. 1E, in CAD⫹/⫹ mice, massive chromatin fragmentation was induced in the thymus, spleen, and bone marrow by
␥-irradiation, and by D-galactosamine/LPS in the liver (septic
shock). In contrast, no in vivo fragmentation was detectable in the
CAD⫺/⫺ mouse. To confirm that the lack of chromatin fragmen-
tation also leads to impaired release of intranuclear components,
DNA laddering was evaluated in plasma from CAD⫹/⫹ and
CAD⫺/⫺ mice 8 h after ␥-irradiation treatment. Again, circulating
fragmented chromatin was detected only in CAD⫹/⫹ mice, demonstrating that in CAD⫺/⫺ mice, extra cellular release of nuclear
content is absent or extremely reduced (Fig. 1F).
The Journal of Immunology
FIGURE 3. CAD⫹/⫹ and CAD⫺/⫺ are equally susceptible to EAE.
EAE was induced with a single injection of myelin oligodendrocyte glycoprotein peptide in CAD⫹/⫹ and CAD⫺/⫺ mice, accompanied by i.p.
injection of Pertussis toxin (200 ng in 200 ␮l PBS) at the time of immunization and 48 h later. Females and males mice were tested separately (A
and B, respectively) and were monitored for at least 3 wk until the onset of
neurological symptoms. Both mouse strains showed similar incidence and
severity of disease (p ⫽ n.s.).
Induction of SLE-like autoimmune disease promotes comparable
increases of Ig levels in both CAD⫹/⫹ and CAD⫺/⫺ mice
CAD⫺/⫺ mice challenged with pristane fail to develop lupus
autoantibodies toward nuclear components but maintain
autoimmunity toward membrane phospholipids
To determine in vivo the relevance of apoptotic chromatin fragmentation and the subsequent nuclear fragmentation, apoptotic
body formation, and release of nuclear contents into the circula-
FIGURE 4. CAD⫹/⫹ and CAD⫺/⫺ mice similarly respond to pristane
treatment by producing immunoglobulins. A total of 27 CAD⫹/⫹ and 24
CAD⫺/⫺ mice were injected with pristane mineral oil to induce lupus-like
autoimmunity in three separate experiments. The result of one experiment
is shown. Similar results were obtained in the other two experiments. Serum Ig levels were tested by ELISA before and 4 mo after injection. Each
line corresponds to an individual mouse. Both CAD⫹/⫹ and CAD⫺/⫺
mice responded by increasing the levels of all Ig isotypes without significant differences, except in IgA levels (p ⫽ 0.014) that were higher in the
CAD⫺/⫺ mice, demonstrating that the lack of CAD did not interfere with
this particular aspect of pristane-induced lupus.
tion, we investigated the occurrence and specificity of pristaneinduced lupus autoantibodies in CAD⫹/⫹ and CAD⫺/⫺ mice.
We first tested anti-chromatin Abs by ELISA in CAD⫹/⫹ and
CAD⫺/⫺ mice 4 mo after pristane injection. Although the levels
of autoantibodies were overall low, although, CAD⫹/⫹ mice
showed a statistically significant increase in anti-chromatin Abs
( p ⫽ 0.0078), while CAD⫺/⫺ mice did not ( p ⫽ 0.9375) (Fig.
5A). As expected, both CAD⫹/⫹ and CAD⫺/⫺ mice were negative for anti-dsDNA Abs (data not shown) (23). To determine
whether the absence of chromatin and nuclear fragmentation impaired the generation of other lupus nuclear-targeted autoantibodies, we used IP (23). Radiolabeled K562 cell extract was incubated
with sera 6 mo after pristane injection. Sera were obtained in three
separate experiments from a total of 27 CAD⫹/⫹ and 24
CAD⫺/⫺ mice. The results showed that 12 of 27 CAD⫹/⫹ mice
but only 3 of 24 CAD⫺/⫺ mice became positive for known lupus
autoantibodies (Fig. 5B, Table II, p ⬍0.05). Autoantibodies toward
nuclear components such as the snRNPs, the pathognomonic target
of lupus autoantibodies, and NF110/90/45 complexes were identified only in CAD⫹/⫹ mice ( p ⬍0.05). In contrast, autoantibodies against cytoplasmic components such as Su and signal recognition particle were generated in both CAD⫺/⫺ and CAD⫹/⫹
mice (Fig. 5B and Table II, p ⫽ n.s.).
To confirm and extend these findings, we performed ANA staining on HEp-2 cells with sera from pristane-injected mice. Nine of
twenty-seven CAD⫹/⫹ mice were positive for ANA, while only
two of the CAD⫺/⫺ mice were positive (Fig. 5C and Table II),
consistently showing a reduction of autoantibodies ( p ⫽ 0.01).
These results confirm the impairment of humoral autoimmunity in
CAD⫺/⫺ mice.
Interestingly, similar to the IP results, ANA staining in the
CAD⫺/⫺ mice showed absence of Abs against nuclear components ( p ⬍0.05) but also a similar prevalence for cytoplasmic targets (Table II and Fig. 5C, p ⫽ n.s).
We have shown that the absence of CAD does not interfere with
membrane phospholipid exposure during cell death (Fig. 1D).
Therefore we tested whether anti-cardiolipin (aCL) Abs, lupus Abs
that target membrane phospholipids exposed during cell death
(25), were still generated in the absence of CAD. Indeed, 4 mo
after pristane injection, both CAD⫹/⫹ and CAD⫺/⫺ mice demonstrated significant increases of aCL levels (Fig. 5D, p ⫽ 0.003,
respectively). Although aCL increased in CAD⫺/⫺ mice, their
levels were lower than in CAD⫹/⫹ mice. Perhaps the absence of
nuclear fragmentation and blebbing partially interferes with CL Ag
recognition but is not sufficient to impair the development of
autoantibodies.
Overall, these results demonstrate that cell death molecular
steps, such as fragmentation of chromatin and phospholipids exposure, interfere with the generation of specific autoantibodies.
Moreover, the absence of autoantibodies to known major nuclear
autoantigens in the CAD⫺/⫺ mice was accompanied by autoimmunity toward cytoplasmic targets, as shown by the generation of
autoantibodies to Su and SRP (Fig. 5, B and C, and Table II).
Both pristane treated CAD⫹/⫹ and CAD⫺/⫺ mice develop
mild kidney disease
We next evaluated renal disease in the pristane model of SLE (23).
Seven months after injection, mice were sacrificed and kidneys
were evaluated for the severity of nephritis and the presence of
immune deposits. Glomerulonephritis was scored according to established guidelines previously published (41).
As shown in Fig. 6A, CAD⫺/⫺ and CAD⫹/⫹ mice scored
similarly (p ⫽ n.s). The intensity of immune deposits was also
very similar (p ⫽ n.s., data not shown). Particularly noteworthy,
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The mineral oil pristane induces an autoimmune syndrome similar
to SLE when injected in healthy mice, accompanied by a B cell
polyclonal activation and generalized increase of Ig levels in all
subclasses (39, 40). Two-month-old CAD⫹/⫹ and CAD⫺/⫺
mice received a single i.p. injection of pristane and were monitored
for several months. Four months after injection, IgG1, IgG2a,
IgG2b, IgG3, IgM, and IgA levels were tested by ELISA. As
shown in Fig. 4, in this experiment, the levels of all Ig subclasses
increased after pristane injection in both CAD⫹/⫹ and CAD⫺/⫺
mice. Although some degree of variability existed from mouse to
mouse in each group, the only statistical difference detectable between the two groups before or after treatment ( p ⬎0.05) was in
IgA levels, which was higher in the CAD⫺/⫺ mice ( p ⫽ 0.014).
Similar results were obtained in the other two experiments. These
results demonstrate that CAD⫺/⫺ mice reacted as expected, with
a B cell polyclonal response to the initial pristane challenge.
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LACK OF CHROMATIN FRAGMENTATION IMPAIRS LUPUS ANA
since we found increased levels of IgA in the CAD⫺/⫺, we also
stained glomeruli for IgA. However, there were comparable IgA
deposits in CAD⫹/⫹ and CAD⫺/⫺ mice (data not shown). Examples of kidney sections from the pristane induced lupus and
spontaneous lupus murine model MRL/lpr are shown in Fig. 6B.
Discussion
We report that mice lacking the capacity of fragmenting chromatin
and nuclei during apoptosis failed to generate ANA, including antichromatin and anti-snRNPs, following induction of an SLE-like
disease.
Our data clearly indicate that CAD⫺/⫺ mice are unable to fragment DNA and nuclei during apoptosis both in vitro and in vivo.
We also found that chromatin is not released systemically in vivo
and in the supernatant of CAD⫺/⫺ cultured cells in vitro. Our
results thus concur with the literature that emphasizes the role of
CAD over that of other nucleases in causing chromatin fragmentation in vivo following a variety of apoptotic stimuli (42). Other
critical steps of the apoptosis process, such as exposure of phospholipids and caspase-3 activation, are however intact in the absence of CAD. Since we tested both immune and nonimmune tissues, we conclude that CAD is systemically required for chromatin
and nuclear fragmentation during apoptosis, indicating that
CAD⫺/⫺ mice are a suitable model to investigate autoimmunity
in the absence of the generation of apoptosis-induced lupus
autoantigens.
We induced lupus by injecting a single i.p. dose of pristane oil
(22). In this experimental model, lupus develops within months
and is characterized by production of ANAs and mild glomerulonephritis (43). Recently, it has been proposed that apoptotic cell
Table II. Nuclear and cytoplasmic Ags after pristane-induced lupus
CAD⫹/⫹
IP
Antigen
Nuclear
Cytosplasmic
NF/snRNP
Su/SRP
Pattern
Nuclear
Cytoplasmic
ANA (1:160)
a
Percentages of positive mice.
b
Statistical significance (p ⬍ 0.05).
c
Not significant.
CAD⫺/⫺
a
12/27 (44.4)
8/27 (29.6)
7/27 (25.9)
9/27 (33.3)
7/27 (25.9)
2/27 (7.4)
3/24 (12.5)b
0/24 (0)b
3/24 (12.5)c
2/24 (8.3)b
0/24 (0)b
2/24 (8.3)c
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FIGURE 5. CAD⫺/⫺ mice failed to develop specific nuclear autoantibodies in the pristane-induced SLE-like autoimmune-disease. The same mice,
shown in Fig. 4, were tested for Ab specificities. Data on individual mice are shown. A, CAD⫹/⫹ mice showed a significant increase of anti-chromatin
Abs, while CAD⫺/⫺ mice did not (p ⫽ 0.0078 vs p ⫽ 0.9375). B, IP of sera from three experiments of pristane-injected CAD ⫹/⫹ and CAD⫺/⫺ mice
are shown. Autoantibodies against snRNPs, Su, and NF 110/90/45 were found in the majority of CAD⫹/⫹ sera, while only three CAD⫺/⫺ mouse had
Abs such as anti-Su and anti-SRP Abs. (ⴱ) indicates sera positive for known lupus Ags. See also Table II. C, ANA from five examples of CAD ⫺/⫺ and
⫹/⫹ mice are shown. Controls are sera of non pristane injected mice. The serum of an ANA positive MRL/lpr mouse was chosen as positive control.
Several sera from CAD⫹/⫹ mice showed presence of ANA, compared with fewer CAD⫺/⫺ sera. Refer to Table II for more details. D, Anti-cardiolipin
Abs were detected by ELISA, with both CAD⫹/⫹ and CAD⫺/⫺ mice demonstrating a significant increase of this specificity 4 mo after pristane injection
(p ⫽ 0.003, respectively).
The Journal of Immunology
triggering of TLR7 and TLR9, due to the lack of circulating Ag,
contributed to the impairment of autoimmunity.
Finally, our study and those on lupus models dependent on impaired clearance of apoptotic cells raise a critical question: is there
a particular cell type or tissue more prone than others to provide
lupus autoantigens? Indirect evidence suggests that B lymphocytes
(46) and macrophages (47) are the “culprits,” and experiments are
underway in our laboratory to determine whether immune cells or
other cell types are the primary suppliers of autoantigens.
In conclusion, our results strongly suggest that during apoptotic
cell death and in the absence of CAD-dependent chromatin and
nuclear fragmentation, specific lupus nuclear autoantibodies do not
develop, indicating that apoptotic cells and their by-products are
indeed a source of lupus autoantigens in vivo. Importantly, the lack
of exposure to major nuclear lupus autoantigens induces the generation of different auto-Ab specificities, especially to cytoplasmic
targets. Our results also show that the absence of chromatin fragmentation specifically interferes with nuclear lupus autoimmunity
but does not interfere with cytoplasmic membrane lupus autoantibodies (aCL) and with other forms of autoimmunity, such as
EAE. Experiments are underway to determine whether apoptotic
Ags specifically generated by the cell death machinery also drive
autoimmunity in spontaneous murine lupus models.
Acknowledgments
We thank Drs. P. Cohen, R. Eisenberg, S. Gallucci, T. Laufer, and
G. Koretzky, for critically reading the manuscript and for their excellent
suggestions. We are grateful to Dr. S. Nagata for providing the invalvable
CAD⫺/⫺ mice. We also thank Dr. G. Wu for providing reagents and his
expertise in the induction and neurological evaluation of
EAE. We are indebted to Dr. M. Gallucci for helping with the statistical
analysis.
Disclosures
The authors have no financial conflict of interest.
death supplies autoantigens because pristane induces cell death of
lymphoid cells in the peritoneal cavity along with proinflammatory
cytokines and secondary necrosis (24).
Following pristane injection, Ig levels increased in both strains,
except for IgA that showed significantly higher levels in the absence of CAD. Nevertheless, the IgA increase did not result in
increased IgA-specific immune complex deposition in the glomeruli (data not shown).
In the present study, the drastic reduction of ANAs in the absence of CAD cannot be ascribed to an impaired production of
Abs. Indeed, we found that CAD⫺/⫺ mice have a functional immune system and produce a normal Ag-dependent humoral immune response. Moreover, they are susceptible to EAE and respond to pristane by increasing serum Ig levels similarly to the
response observed in ⫹/⫹ mice. In contrast, we showed that
CAD⫺/⫺ mice also lack the capability of generating blebs (20)
and bodies upon induction of apoptotic cell death. These subcellular structures can concentrate lupus autoantigens and are the
most suitable material to be phagocytosed by dendritic cells (1,
13).
The absence of CAD led to the production of autoantibodies
with cytoplasmic specificity as demonstrated by the immunoprecipitation studies and the ANA patterns. The lack of auto-Ab and
their skewing toward a cytoplasmic repertoire is in agreement with
the results obtained by Christensen et al. in lupus-prone mice that
have been rendered TLR7- or TLR9-deficient (44, 45). In these
mice, the generation of anti-snRNPs and anti-chromatin autoantibodies was dependent on the TLR triggered by the specific autoantigen (45). It is therefore possible that, in our model, a reduced
References
1. Casciola-Rosen, L. A., G. Anhalt, and A. Rosen. 1994. Autoantigens targeted in
systemic lupus erythematosus are clustered in two populations of surface structures on apoptotic keratinocytes. J. Exp. Med. 179: 1317–1330.
2. Mevorach, D., J. L. Zhou, X. Song, and K. B. Elkon. 1998. Systemic exposure to
irradiated apoptotic cells induces autoantibody production. J. Exp. Med. 188:
387–392.
3. Cohen, P. L., R. Caricchio, V. Abraham, T. D. Camenisch, J. C. Jennette,
R. A. Roubey, H. S. Earp, G. Matsushima, and E. A. Reap. 2002. Delayed apoptotic cell clearance and lupus-like autoimmunity in mice lacking the c-mer
membrane tyrosine kinase. J. Exp. Med. 196: 135–140.
4. Korb, L. C., and J. M. Ahearn. 1997. C1q binds directly and specifically to
surface blebs of apoptotic human keratinocytes: complement deficiency and systemic lupus erythematosus revisited. J. Immunol. 158: 4525– 4528.
5. Bickerstaff, M. C., M. Botto, W. L. Hutchinson, J. Herbert, G. A. Tennent,
A. Bybee, D. A. Mitchell, H. T. Cook, P. J. Butler, M. J. Walport, and
M. B. Pepys. 1999. Serum amyloid P component controls chromatin degradation
and prevents antinuclear autoimmunity. Nat. Med. 5: 694 – 697.
6. Napirei, M., H. Karsunky, B. Zevnik, H. Stephan, H. G. Mannherz, and
T. Moroy. 2000. Features of systemic lupus erythematosus in Dnase1-deficient
mice. Nat. Genet. 25: 177–181.
7. Gensler, T. J., M. Hottelet, C. Zhang, S. Schlossman, P. Anderson, and P. J. Utz.
2001. Monoclonal antibodies derived from BALB/c mice immunized with apoptotic Jurkat T cells recognize known autoantigens. J. Autoimmun. 16: 59 – 69.
8. Baumann, I., W. Kolowos, R. E. Voll, B. Manger, U. Gaipl, W. L. Neuhuber,
T. Kirchner, J. R. Kalden, and M. Herrmann. 2002. Impaired uptake of apoptotic
cells into tingible body macrophages in germinal centers of patients with systemic
lupus erythematosus. Arthritis Rheum. 46: 191–201.
9. Caricchio, R., L. Mcphie, and P. Cohen. 2003. UV-B radiation-induce cell death:
critical role of UV dose in inflammation and lupus autoantigen redistribution.
J. Immunol. 171: 5778 –5786.
10. Pisetsky, D. S. 2004. DNA as a marker of cell death in systemic lupus erythematosus. Rheum. Dis. Clin. N. Am. 30: 575–587, x.
11. Wyllie, A. H., J. F. Kerr, and A. R. Currie. 1980. Cell death: the significance of
apoptosis. Int. Rev. Cytol. 68: 251–306.
12. Sakahira, H., M. Enari, Y. Ohsawa, Y. Uchiyama, and S. Nagata. 1999. Apoptotic
nuclear morphological change without DNA fragmentation. Curr. Biol. 9:
543–546.
Downloaded from http://www.jimmunol.org/ by guest on August 2, 2017
FIGURE 6. CAD⫹/⫹ and CAD⫺/⫺ showed similar kidney damage.
Glomerulonephritis was assessed as previously described in the material
and methods. A, H&E stained sections from three groups of CAD⫹/⫹ and
CAD⫺/⫺ mice were scored showing comparable kidney damage. Concomitantly, the kidney involvement in the pristane model of lupus was
limited for all groups and the severity of the disease was mild. B, Frozen
sections of kidneys from pristane-treated CAD⫹/⫹ and CAD⫺/⫺ mice
were stained for immune complex deposition using an FITC-conjugated
goat anti-mouse IgG (heavy and light) Ab. As positive control, kidney
sections from an old MRL/lpr mouse that spontaneously developed autoimmunity and kidney disease were used. As negative control, an agematched untreated C57BL/6 mouse was used. H&E staining on formalin
fixed kidney sections showed as well that the absence of CAD and chromatin fragmentation did not interfere with the development of renal
disease.
7965
7966
LACK OF CHROMATIN FRAGMENTATION IMPAIRS LUPUS ANA
31. Chan, O., M. P. Madaio, and M. J. Shlomchik. 1997. The roles of B cells in
MRL/lpr murine lupus. Ann. NY Acad. Sci. 815: 75– 87.
32. Ogasawara, J., R. Watanabe-Fukunaga, M. Adachi, A. Matsuzawa, T. Kasugai,
Y. Kitamura, N. Itoh, T. Suda, and S. Nagata. 1993. Lethal effect of the anti-Fas
antibody in mice. Nature 364: 806 – 809.
33. Caricchio, R., E. A. Reap, and P. L. Cohen. 1998. Fas/Fas ligand interactions are
involved in ultraviolet-B-induced human lymphocyte apoptosis. J. Immunol. 161:
241–251.
34. Martin, S. J., C. P. Reutelingsperger, A. J. McGahon, J. A. Rader,
R. C. van Schie, D. M. LaFace, and D. R. Green. 1995. Early redistribution of
plasma membrane phosphatidylserine is a general feature of apoptosis regardless
of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. J. Exp.
Med. 182: 1545–1556.
35. McIlroy, D., M. Tanaka, H. Sakahira, H. Fukuyama, M. Suzuki, K. Yamamura,
Y. Ohsawa, Y. Uchiyama, and S. Nagata. 2000. An auxiliary mode of apoptotic
DNA fragmentation provided by phagocytes. Genes Dev. 14: 549 –558.
36. Fadok, V. A., and G. Chimini. 2001. The phagocytosis of apoptotic cells. Semin.
Immunol. 13: 365–372.
37. Casciola-Rosen, L., D. W. Nicholson, T. Chong, K. R. Rowan, N. A. Thornberry,
D. K. Miller, and A. Rosen. 1996. Apopain/CPP32 cleaves proteins that are
essential for cellular repair: a fundamental principle of apoptotic death. J. Exp.
Med. 183: 1957–1964.
38. Gold, R., C. Linington, and H. Lassmann. 2006. Understanding pathogenesis and
therapy of multiple sclerosis via animal models: 70 years of merits and culprits
in experimental autoimmune encephalomyelitis research. Brain 129: 1953–1971.
39. Hamilton, K. J., M. Satoh, J. Swartz, H. B. Richards, and W. H. Reeves. 1998.
Influence of microbial stimulation on hypergammaglobulinemia and autoantibody production in pristane-induced lupus. Clin. Immunol. Immunopathol. 86:
271–279.
40. Naim, J. O., M. Satoh, N. A. Buehner, K. M. Ippolito, H. Yoshida, D. Nusz,
L. Kurtelawicz, S. F. Cramer, and W. H. Reeves. 2000. Induction of hypergammaglobulinemia and macrophage activation by silicone gels and oils in female
A.SW mice. Clin. Diagn. Lab. Immunol. 7: 366 –370.
41. Sekiguchi, D. R., S. M. Jainandunsing, M. L. Fields, M. A. Maldonado,
M. P. Madaio, J. Erikson, M. Weigert, and R. A. Eisenberg. 2002. Chronic graftversus-host in Ig knockin transgenic mice abrogates B cell tolerance in antidouble-stranded DNA B cells. J. Immunol. 168: 4142– 4153.
42. Nagata, S. 2005. DNA degradation in development and programmed cell death.
Annu. Rev. Immunol. 23: 853– 875.
43. Kuroda, Y., D. C. Nacionales, J. Akaogi, W. H. Reeves, and M. Satoh. 2004.
Autoimmunity induced by adjuvant hydrocarbon oil components of vaccine.
Biomed. Pharmacother. 58: 325–337.
44. Christensen, S. R., J. Shupe, K. Nickerson, M. Kashgarian, R. A. Flavell, and
M. J. Shlomchik. 2006. Toll-like receptor 7 and TLR9 dictate autoantibody specificity and have opposing inflammatory and regulatory roles in a murine model of
lupus. Immunity 25: 417– 428.
45. Christensen, S. R., M. Kashgarian, L. Alexopoulou, R. A. Flavell, S. Akira, and
M. J. Shlomchik. 2005. Toll-like receptor 9 controls anti-DNA autoantibody production in murine lupus. J. Exp. Med. 202: 321–331.
46. Hanayama, R., M. Tanaka, K. Miyasaka, K. Aozasa, M. Koike, Y. Uchiyama,
and S. Nagata. 2004. Autoimmune disease and impaired uptake of apoptotic cells
in MFG-E8-deficient mice. Science 304: 1147–1150.
47. Denny, M. F., P. Chandaroy, P. D. Killen, R. Caricchio, E. E. Lewis,
B. C. Richardson, K. D. Lee, J. Gavalchin, and M. J. Kaplan. 2006. Accelerated
macrophage apoptosis induces autoantibody formation and organ damage in systemic lupus erythematosus. J. Immunol. 176: 2095–2104.
Downloaded from http://www.jimmunol.org/ by guest on August 2, 2017
13. Frisoni, L., L. McPhie, L. Colonna, U. Sriram, M. Monestier, S. Gallucci, and
R. Caricchio. 2005. Nuclear autoantigen translocation and autoantibody opsonization lead to increased dendritic cell phagocytosis and presentation of nuclear
antigens: a novel pathogenic pathway for autoimmunity? J. Immunol. 175:
2692–2701.
14. Amoura, Z., S. Koutouzov, and J. C. Piette. 2000. The role of nucleosomes in
lupus. Curr. Opin. Rheumatol. 12: 369 –373.
15. Lau, C. M., C. Broughton, A. S. Tabor, S. Akira, R. A. Flavell, M. J. Mamula,
S. R. Christensen, M. J. Shlomchik, G. A. Viglianti, I. R. Rifkin, and
A. Marshak-Rothstein. 2005. RNA-associated autoantigens activate B cells by
combined B cell antigen receptor/toll-like receptor 7 engagement. J. Exp. Med.
202: 1171–1177.
16. Boule, M. W., C. Broughton, F. Mackay, S. Akira, A. Marshak-Rothstein, and
I. R. Rifkin. 2004. Toll-like receptor 9-dependent and -independent dendritic cell
activation by chromatin-immunoglobulin G complexes. J. Exp. Med. 199:
1631–1640.
17. Enari, M., H. Sakahira, H. Yokoyama, K. Okawa, A. Iwamatsu, and S. Nagata.
1998. A caspase-activated DNase that degrades DNA during apoptosis, and its
inhibitor ICAD. Nature 391: 43–50.
18. Kawane, K., H. Fukuyama, H. Yoshida, H. Nagase, Y. Ohsawa, Y. Uchiyama,
K. Okada, T. Iida, and S. Nagata. 2003. Impaired thymic development in mouse
embryos deficient in apoptotic DNA degradation. Nat. Immunol. 4: 138 –144.
19. Nagata, S., H. Nagase, K. Kawane, N. Mukae, and H. Fukuyama. 2003. Degradation of chromosomal DNA during apoptosis. Cell Death Differ. 10: 108 –116.
20. Zhang, J., X. Wang, K. E. Bove, and M. Xu. 1999. DNA fragmentation factor
45-deficient cells are more resistant to apoptosis and exhibit different dying morphology than wild-type control cells. J. Biol. Chem. 274: 37450 –37454.
21. McIlroy, D., H. Sakahira, R. V. Talanian, and S. Nagata. 1999. Involvement of
caspase 3-activated DNase in internucleosomal DNA cleavage induced by diverse apoptotic stimuli. Oncogene 18: 4401– 4408.
22. Satoh, M., and W. H. Reeves. 1994. Induction of lupus-associated autoantibodies
in BALB/c mice by intraperitoneal injection of pristane. J. Exp. Med. 180:
2341–2346.
23. Satoh, M., A. Kumar, Y. S. Kanwar, and W. H. Reeves. 1995. Anti-nuclear
antibody production and immune-complex glomerulonephritis in BALB/c mice
treated with pristane. Proc. Natl. Acad. Sci. USA 92: 10934 –10938.
24. Calvani, N., R. Caricchio, M. Tucci, E. S. Sobel, F. Silvestris, P. Tartaglia, and
H. B. Richards. 2005. Induction of apoptosis by the hydrocarbon oil pristane:
implications for pristane-induced lupus. J. Immunol. 175: 4777– 4782.
25. Nicolo, D., B. I. Goldman, and M. Monestier. 2003. Reduction of atherosclerosis
in low-density lipoprotein receptor-deficient mice by passive administration of
antiphospholipid antibody. Arthritis Rheum. 48: 2974 –2978.
26. Rodriguez, I., K. Matsuura, C. Ody, S. Nagata, and P. Vassalli. 1996. Systemic
injection of a tripeptide inhibits the intracellular activation of CPP32-like proteases in vivo and fully protects mice against Fas-mediated fulminant liver destruction and death. J. Exp. Med. 184: 2067–2072.
27. Reap, E. A., D. Leslie, M. Abrahams, R. A. Eisenberg, and P. L. Cohen. 1995.
Apoptosis abnormalities of splenic lymphocytes in autoimmune lpr and gld mice.
J. Immunol. 154: 936 –943.
28. Caricchio, R., D. Kovalenko, W. K. Kaufmann, and P. L. Cohen. 1999. Apoptosis
provoked by the oxidative stress inducer menadione (Vitamin K(3)) is mediated
by the Fas/Fas ligand system. Clin. Immunol. 93: 65–74.
29. Racke, M. K. 2001. Experimental Autoimmune Encephalomyelitis (EAE). John
Wiley and Sons, Inc, New York.
30. Chan, O. T., M. P. Madaio, and M. J. Shlomchik. 1999. B cells are required for
lupus nephritis in the polygenic: Fas-intact MRL model of systemic autoimmunity. J. Immunol. 163: 3592–3596.