The Combination of Apoptotic U937 Cells
and Lupus IgG Is a Potent IFN- α Inducer
Ullvi Båve, Gunnar V. Alm and Lars Rönnblom
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References
J Immunol 2000; 165:3519-3526; ;
doi: 10.4049/jimmunol.165.6.3519
http://www.jimmunol.org/content/165/6/3519
The Combination of Apoptotic U937 Cells and Lupus IgG Is a
Potent IFN-␣ Inducer1
Ullvi Båve,2* Gunnar V. Alm,† Lars Rönnblom*
S
ystemic lupus erythematosus (SLE)3 is a multisystem autoimmune disease characterized by production of autoantibodies against DNA and nucleic acid-associated proteins
(1). The type I IFN system may be important in the autoimmune
process because increased levels of circulating IFN-␣ and of IFN␣-inducible cellular proteins are found in SLE patients and correlate to disease activity (2– 8). Furthermore, patients with nonautoimmune disorders treated with IFN-␣ frequently develop
antinuclear Abs, Abs to native DNA, and occasionally SLE (9 –
12). Interestingly, we recently identified an IFN-␣-inducing factor
(SLE-IIF) in sera from SLE patients, especially those with active
disease (13). Given the important immunoregulatory functions of
type I IFN (14 –19), the SLE-IIF could impair induction of selftolerance and promote autoimmunity via produced IFN-␣. Such an
action is rendered even more likely by the fact that the SLE-IIF
appears to selectively activate the natural IFN-producing cells
(natural IPC) (13), which have a phenotype resembling that of
immature dendritic cells (DC) of the DC2 type (13, 20 –22) that are
potent stimulators of immune responses (23).
The SLE-IIF was shown to consist of complexes of anti-DNA
Abs and DNA (13), the latter resembling hypomethylated immunostimulatory (is) DNA in function (24). The DNA component
may be derived from apoptotic cells, because SLE patients have
*Section of Rheumatology, Department of Medical Sciences, Uppsala University; and
†
Section of Immunology, Department of Veterinary Microbiology, Swedish University of Agricultural Sciences, Uppsala, Sweden
Received for publication November 8, 1999. Accepted for publication July 5, 2000.
been reported to have both increased apoptosis and reduced clearance of apoptotic cell material (25–28). In the present study, we
therefore investigated whether apoptotic cells, together with IgG
from SLE patients, could act as an IFN-␣ inducer in normal PBMC
in vitro. We found that apoptotic cells of the U937 myeloid leukemia cell line could induce IFN-␣ production in PBMC when
combined with IgG from SLE patients. The ability of IgG from
different SLE patients to promote IFN-␣ induction by apoptotic
U937 cells appeared related to presence of anti-ribonucleoprotein
(RNP) Abs, but not to anti-DNA Abs. This mechanism for induction of IFN-␣ production may explain the IFN-␣ production seen
in SLE patients in vivo and could be important in the pathogenesis
of the disease.
Materials and Methods
Patients and Controls
A total of 22 SLE patients (20 female and 2 male) with a median age of
42.5 years (range 14 – 68 years) and a mean duration of disease of 10.5
years (range 1– 49 years) were included in the study. The median American
College of Rheumatology index (29) for the patients was 6 (range 4 –10).
Disease activity was measured by the SLE disease activity index (SLEDAI) (30), where complement levels and anti-DNA Abs were excluded.
Citrated plasma samples were obtained, converted to serum by addition of
1 M CaCl2, and stored at – 80°C. A large plasma sample was collected by
plasmapheresis of a 16-year-old female patient (SLE 1), with a SLEDAI
score of 10. Plasma samples from four normal blood donors, median age 28
years (range 20 –31 years), served as controls. The study protocol was
approved by the Committee of Ethics, Faculty of Medicine, Uppsala
University.
Determination of autoantibodies
1
This work was supported by grants from the Tore Nilson Foundation, the 80 Years
Foundation of King Gustaf V, the Swedish Rheumatism Foundation, the Tore and
Wera Cornell Foundation, and the Swedish Medical Research Council.
2
Address correspondence and reprint requests to Dr. Ullvi Båve, Immunology (V),
Biomedical Center, P.O. Box 588, 75123 Uppsala, Sweden. E-mail address:
[email protected]
3
Abbreviations used in this paper: SLE, systemic lupus erythematosus; CL, cardiolipin; DELFIA, dissociation-enhanced lanthanide fluoroimmunoassay; DC, dendritic
cell; IIF, IFN-␣-inducing factor; IPC, IFN-␣-producing cells; isDNA, immunostimulatory DNA; SLEDAI, SLE disease activity index. RNP, ribonucleoprotein; Sm,
Smith; SSA and B, Sjögrens syndrome Ag A and B; PI, propidium iodide.
Copyright © 2000 by The American Association of Immunologists
Specific autoantibodies directed toward Smith (Sm), RNP or Sjögrens Syndrome antigens A and B (SSA and SSB, also termed Ro and La) (1), were
determined by investigations with immunodiffusion (Auto I.D. plates; Immuno Concepts, Sacramento, CA) yielding immunological identity reactions with predefined autoantibody containing samples. IgG anticardiolipin (anti-CL) Abs were determined using a commercial ELISA
(Autozyme; Cambridge Life Sciences, Ely, Cambridgeshire, U.K.). Abs
against dsDNA were detected by indirect immunofluorescence using
Crithidia luciliae (Immuno Concepts) or when indicated by an anti-dsDNA
Ab ELISA kit (Dako, Glostrup, Denmark).
0022-1767/00/$02.00
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
Patients with active systemic lupus erythematosus (SLE) have signs of an ongoing IFN-␣ production, that may be of pathogenic
significance in the disease. We previously showed that SLE patients have an IFN-␣-inducing factor in blood, probably consisting
of complexes containing anti-DNA Abs and immunostimulatory DNA. The DNA component could be derived from apoptotic cells,
because SLE patients have been reported to have both increased apoptosis and reduced clearance of apoptotic cell material. In the
present study, we therefore investigated whether apoptotic cells, together with IgG from SLE patients, could act as an IFN-␣
inducer in normal PBMC in vitro. We found that apoptotic cells of the myeloid leukemia cell line U937 as well as four other cell
lines (MonoMac6, H9, Jurkat, U266) could induce IFN-␣ production in PBMC when combined with IgG from SLE patients. The
IFN-␣ production by PBMC was much enhanced when PBMC were costimulated by IFN-␣2b. The ability of IgG from different
SLE patients to promote IFN-␣ induction by apoptotic U937 cells was associated with the presence of anti-ribonucleoprotein Abs,
but not clearly with occurrence of anti-DNA Abs. These results suggest that apoptotic cells in the presence of autoantibodies can
cause production of a clearly immunostimulatory cytokine, which is IFN-␣. This mechanism for induction of IFN-␣ production
could well be operative also in vivo, explain the IFN-␣ production seen in SLE patients, and be important in the pathogenesis of
SLE. The Journal of Immunology, 2000, 165: 3519 –3526.
3520
APOPTOTIC U937 CELLS COMBINED WITH SLE-IgG INDUCE IFN-␣
Preparation of IgG and removal of anti-DNA Ab
Sera were prefiltered by using 0.45-␮m filters (Acrodisc; Gelman Sciences,
Ann Arbor, MI) and then treated for 1 h at 37°C with equal volumes of
DNAse I (2000 U/ml; Boehringer Mannheim, Mannheim, Germany) in 100
mM Tris-HCl (pH 7.5) and 10 mM MnCl2 to eliminate endogenous DNA.
Subsequently, IgG was purified on protein G Sepharose (Amersham Pharmacia Biotech, Uppsala, Sweden) as recommended by the manufacturer.
The IgG containing eluates were dialyzed against RPMI 1640 medium
(ICN Biomedical, Costa Mesa, CA) supplemented with penicillin (60 ␮g/
ml), streptomycin (100 ␮g/ml), L-glutamine (2 mM), and HEPES (20 mM)
and was used at a concentration of 1 mg/ml in the cultures.
Abs against dsDNA and ssDNA were removed by separation of IgG
from patient SLE-1 on dsDNA or ssDNA cellulose columns (Worthington,
Freehold, NJ) using uncoupled cellulose columns as control (31). The effluents were desalted and transferred to RPMI 1640 medium, supplemented
as described above, using PD-10 columns (Amersham Pharmacia Biotech).
Human monoclonal anti-dsDNA and anti-ss/dsDNA Abs (MER-2 and
MER-3; Serotec, Oxford, U.K) were dialyzed against RPMI 1640 medium
and used in cultures at concentrations of 7.5 ␮g/ml and 2.5 ␮g/ml,
respectively.
Culture and treatment of cell lines
Immunoassay for IFN-␣
The levels of IFN-␣ in culture supernatants were determined by dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) as described (34),
with modifications. Briefly, microtiter plates were coated with the antiIFN-␣ mAb LT27:293, which detects the majority of IFN-␣ subtypes but
not the IFN-␣2b used for costimulation in the IFN-␣ induction cultures.
Samples and standard were then coincubated with the europium-labeled
LT27:297 anti-IFN-␣ mAb for 1 h at 37°C in the LT27:293-coated immunoplates. The detection level was 2 U/ml. The IFN-␣ standard was calibrated against the National Institutes of Health reference leukocyte IFN-␣
GA-23-902-530.
Statistics
Data are expressed as means ⫾ SD. The significance of differences was
determined by the Wilcoxon signed rank sum test or the Mann-Whitney U
tests as indicated.
Results
Apoptotic U937 cells combined with either SLE serum or IgG
induce IFN-␣ production in PBMCs
We asked if apoptotic U937 cells alone, or when combined with
SLE sera, could trigger IFN-␣ production in normal PBMCs. The
latter were cocultured for 24 h with UV-treated or control-untreated U937 cells and sera from 22 SLE patients, and the IFN-␣
levels were then measured.
A total of 14 of 22 SLE sera alone induced a production of ⬎25
U/ml IFN-␣ in PBMC (Table I), which was in accordance with
earlier findings (13). Apoptotic U937 cells combined with SLE
sera increased the IFN-␣ production significantly ( p ⬍ 0.001),
while untreated U937 cells with the same SLE sera had a much
smaller stimulatory effect ( p ⬍ 0.05). Normal control sera could
not induce IFN-␣ production irrespective of addition of apoptotic
U937 cells or not (results not shown). These results and our previous observations (13, 24) suggested the possibility that autoantibodies from SLE sera together with apoptotic cells are capable of
inducing IFN-␣.
Herpes simplex virus
The HSV was prepared and UV inactivated as described before (13) and
used as a control IFN-␣ inducer at a final concentration of 2 ⫻ 107 PFU/ml
in the cultures.
Table I. Sera from SLE patients together with UV-treated U937 cells
induce IFN-␣ production in normal PBMCa
Preparation and culture of PBMC
Human PBMCs were prepared by Ficoll-Hypaque (Pharmacia, Uppsala,
Sweden) density gradient centrifugation of buffy coats from normal blood
donors. The cells were washed in PBS four times and stored frozen at
– 80°C in FCS containing 10% DMSO. Before using, the PBMCs were
thawed rapidly, washed twice in RPMI 1640 medium, and cocultured at
2 ⫻ 106 cells/ml with U937 cells (0.5 ⫻ 106 cells/ml) in RPMI 1640
medium supplemented as above, but with 3.75% FCS and 500 U/ml recombinant IFN-␣2b (Intron-A; Schering-Plough, Bloomfield, NJ), if not
otherwise indicated. Triplicate cultures with final volumes of 100 ␮l/well
in 96-well round-bottom plates (Nunc) were incubated for 24 h at 37°C and
7% CO2. The PBMCs were initially selected for a good IFN-␣ production
in response to HSV and SLE serum.
Apoptosis assays
Apoptosis was detected by annexin V or TUNEL staining. In brief, the
former was performed by mixing 50 ␮l U937 cells (1 ⫻ 106 cells/ml) with
2.5 ␮l FITC-labelled recombinant annexin V and 5 ␮l propidium iodide
(50 ␮g/ml). After 15 min incubation in the dark at room temperature, 400
␮l of annexin V binding buffer (10 mM Hepes/NaOH, pH 7.4, 140 mM
NaCl, 2.5 mM CaCl2) was added. The TUNEL assay was done using the
Controls
Total IFN-␣ production
Mean net increasec
No. of sera inducing
IFN-␣ productiond
b
Untreated
U937
Cells
17 (2–1103) 35 (2–1064)
26 ⫾ 53
16/22
UV-Treated
U937
Cells
132 (2–2662)
311 ⫾ 500
19/22
a
Untreated or UV-treated (60 mJ) U937 cells were first precultured for 4 h and
then cultured for 24 h with IFN-␣2b (500 U/ml)-costimulated normal PBMC in the
presence of 12.5% serum derived from SLE patients (n ⫽ 22). Controls denote PBMC
cultured with SLE sera, without U937 cells.
b
The figures represent median and range (U/ml) of produced IFN-␣ measured by
immunoassay. Representative results from one of three experiments.
c
Net increase of IFN-␣ levels (mean ⫾ SD; U/ml) caused by addition of untreated
or UV-treated U937 cells compared to controls. The combination of SLE sera and
UV-treated U937 cells induced significantly higher IFN-␣ levels than SLE-sera and
untreated U937 cells ( p ⬍ 0.0005) and controls ( p ⬍ 0.001), using Wilcoxon signed
rank sum test. Untreated U937 cells caused a small increase in IFN-␣ levels compared
to controls ( p ⬍ 0.05).
d
Fourteen of 22 sera induced ⬎25 U/ml IFN-␣ alone, without addition of U937
cells. The proportions of sera given in the table (16/22 and 19/22) represent the
number of SLE sera where addition of untreated or UV-treated U937 cells increased
the IFN-␣ production beyond that caused by serum alone.
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The monocytic cell lines U937 and MonoMac6, the T cell lines Jurkat and
H9, and the B cell line U266 were cultured in RPMI 1640 medium supplemented with 5% FCS (10% for U266) (Myoclone; Life Technologies,
Paisley, U.K.), penicillin (60 ␮g/ml), streptomycin (100 ␮g/ml), L-glutamine (2 mM), and HEPES (20 mM), at 37°C in 7% CO2. The cells were
treated at 1 ⫻ 106 cells/ml by UV light (254 nm, 60 mJ). The U937 cells
were also treated by 12 ␮M etoposide (Sigma, St. Louis, MO) for 3 or 6 h
or by 1 ␮g/ml anti-Fas mAb (clone CH-11; Immunotech, Marseilles,
France) for 1 h. Cells treated with etoposide and anti-Fas mAbs were then
washed once. The treated U937 cells were then cultured for 4 or 24 h (UV
light), for 1 or 18 h (etoposide), and for 3 or 21 h (anti-Fas). The other cell
lines were cultured for 4 h after UV light treatment. For all cultures, 96well round-bottom plates (Nunclon; Nunc, Roskilde, Denmark) were used.
The final concentration of U937 cells used in the cultures was 0.5 ⫻ 106
cells/ml, which was optimal for IFN-␣ induction.
The irreversible caspase inhibitor zVAD-fmk (Calbiochem, Cambridge,
MA) was used at a concentration of 50 ␮M to inhibit apoptosis in some
experiments.
No mycoplasma could be detected in any of the cell lines by staining
with bisbenzimide (Hoechst no. 33258; Sigma). The U937 cells were also
negative when assessed by PCR using mycoplasma group-specific primers
complementary to the 16S rRNA genes (32) or by semi-nested PCR using
primers complementary to universal regions of bacterial 16S rRNA genes
(33).
APO-Direct kit according to the manufacturer’s description, detecting
DNA breaks by incorporation of FITC-labeled dUTP and total DNA by
propidium iodide (PI) staining. All apoptosis assay reagents were obtained
from Pharmingen (San Diego, CA). Analysis of stained cells were performed by a FACScan flow cytometer and the CellQuest software (Becton
Dickinson, San Jose, CA).
The Journal of Immunology
3521
Costimulatory activity of IFN-␣2b
In the present investigation, IFN-␣2b was added to the cultures to
enhance the IFN-␣ production of PBMCs. The IFN-␣2b increased
IFN-␣ production by PBMCs induced by UV-treated U937 cells
and SLE-IgG in a dose-dependent manner (Fig. 3). The increase in
IFN-␣ production was 5-fold in cultures with 500 U/ml of IFN␣2b compared to cultures without IFN-␣2b. Therefore, this IFN␣2b concentration was used in all PBMC cultures to achieve maximal IFN-␣ production. The cytokines GM-CSF and IFN-␥ that in
other systems had costimulatory activity (35) were without costimulatory effect either alone or in combination with IFN-␣ (results not shown). It should be noted that the immunoassay for
IFN-␣ does not detect the added costimulatory IFN-␣2b.
Inhibition of apoptosis in U937 cells reduces their capacity to
induce IFN-␣ production
To verify that U937 cells had to be apoptotic to induce IFN-␣
production, the caspase inhibitor zVAD-fmk was added to the cells
before apoptotic treatment. By adding zVAD-fmk to UV light or
etoposide-treated U937 cells, the level of apoptosis (annexin Vpositive and PI-negative cells) was reduced by 90% and 94%, respectively (Fig. 4A). In contrast, zVAD-fmk did not affect the level
of annexin V-positive and PI-negative staining in U937 cells
treated with anti-Fas, but did decrease the number of apoptotic
cells determined by the TUNEL technique (Fig. 4B).
With regard to the IFN-␣ production, zVAD-fmk markedly inhibited the ability of U937 cells treated by UV, etoposide, or antiFas to induce IFN-␣ production in PBMC in the presence of SLEIgG by 86%, 86%, and 55%, respectively (Fig. 2). In contrast, the
zVAD-fmk did not inhibit the HSV-induced IFN-␣ production in
PBMC (results not shown).
Other autoantibodies than anti-dsDNA Abs are required for
apoptotic U937 cells to induce IFN-␣ production in PBMCs
The combination of anti-dsDNA Abs and DNA is required for the
ability of SLE sera to induce IFN-␣ production in normal PBMCs
in vitro (13, 24). Therefore, we asked whether anti-dsDNA Abs in
SLE-IgG were also necessary for apoptotic U937 cells to induce
IFN-␣ production.
Purified SLE-IgG from patients with ⱖ40 IU/ml anti-dsDNA
Abs, together with apoptotic U937 cells, induced IFN-␣ production in normal PBMCs (Table IV). However, IgG from one patient
without significant levels of anti-DNA Abs (⬍8 IU/ml) was also
stimulatory. In this small patient population, the IFN-␣-inducing
capacity of IgG from patients with active disease as measured by
SLEDAI score was higher compared to IgG from patients in remission ( p ⫽ 0.014, Mann-Whitney U test).
To further demonstrate that anti-DNA Abs were not required for
the IFN-␣ production induced by apoptotic U937 cells, SLE-IgG
was passed over dsDNA columns, ssDNA columns, or both. In all
cases, this resulted in ⬃95% depletion of anti-dsDNA Abs (results
not shown). Still, such SLE-IgG had an intact ability to cause
IFN-␣ synthesis together with UV-treated U937 cells (Fig. 1). Furthermore, monoclonal anti-dsDNA and anti-ssDNA Abs, in the
presence of apoptotic U937 cells, were unable to induce IFN-␣
(results not shown). Consequently, anti-DNA Abs are not required
for the induction of IFN-␣ synthesis in the present experimental
system. Therefore, we attempted to clarify whether other autoantibodies in SLE patients were involved in the induction of IFN-␣
production in the presence of apoptotic U937 cells. This was done
by comparing the ability of SLE sera (same as in Table I) with or
without a certain Ab to induce IFN-␣ production in normal PBMC,
either alone or together with UV-treated U937 cells. The IFN-␣
levels caused by SLE sera alone were subtracted from the IFN-␣
levels caused by the combination of sera and UV-treated cells.
This was done to remove the impact of the previously described
Table II. The combination of apoptotic U937 cells and IgG from SLE patients induce production of IFN-␣a
Culture Supplement
Treatments of U937
None
UV (60 mJ)
Anti-Fas Ab
Etoposide
b
Medium
Normal IgG
SLE-IgG 1
HSV
5⫾8
20 ⫾ 18
17 ⫾ 19
11 ⫾ 2
4⫾5
13 ⫾ 6
11 ⫾ 7
25 ⫾ 17
53 ⫾ 54
1180 ⫾ 219
1147 ⫾ 165
565 ⫾ 136
3485 ⫾ 955
2035 ⫾ 530
2740 ⫾ 935
4060 ⫾ 458
c
a
Normal PBMC were cultured in triplicates for 24 h with U937 cells, together with either medium, normal IgG, SLE-IgG,
or HSV. Levels of IFN-␣ in culture supernatants were then determined by immunoassay.
b
The U937 cells were treated with UV light, anti-Fas Abs for 1 h, or etoposide for 6 h and then cultured for 24 h from start
of apoptotic treatment, before addition of PBMC (see Materials and Methods).
c
The figures represent produced IFN-␣ (U/ml; means ⫾ SD). Representative results from one of three experiments.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
We then investigated whether the component in SLE sera that
triggered IFN-␣ production in combination with apoptotic U937
cells consisted of Ig. The SLE sera were therefore DNAse treated,
and IgG were subsequently purified on a protein G column. This
procedure also had the advantage of destroying the DNA that is an
essential part of the endogenous IFN-␣ inducer in SLE (13). In
addition to UV light, we used etoposide and anti-Fas Abs as inducers of apoptosis in U937 cells. Purified SLE IgG, but not control IgG, in combination with all three types of apoptotic U937
cells resulted in a strong IFN-␣ production in normal PBMC (Table II). The IFN-␣ levels attained were comparable to those caused by
an optimal concentration of the potent viral IFN-␣ inducer HSV.
As controls, apoptotic U937 cells were shown to have no significant effect on HSV-induced IFN-␣ response in PBMC (Table
II). The protein G column effluent was unable to induce IFN-␣
together with apoptotic U937 cells (Fig. 1). Furthermore, viable or
apoptotic U937 cells alone were unable to produce IFN-␣ upon
addition of SLE-IgG, HSV, or IFN-␣2b (results not shown). PBMCs alone, without addition of U937 cells, produced little or no
IFN-␣, regardless of the presence of SLE-IgG (Fig. 2).
Taken together, our data clearly demonstrate that the IFN-␣inducing property was restricted to the combination of SLE-IgG
and apoptotic U937 cells. We further examined whether the ability
to induce IFN-␣ production was unique to U937 cells or present
also in other cell lines. As shown in Table III, MonoMac6, H9,
Jurkat, and U266 cells caused IFN-␣ production in PBMC when
combined with SLE IgG. Furthermore, all cell lines, except H9,
induced more IFN-␣ when treated with UV light.
3522
APOPTOTIC U937 CELLS COMBINED WITH SLE-IgG INDUCE IFN-␣
Table III. Cell lines inducing IFN-␣ production by PBMC in presence
of SLE IgG
IFN-␣ Production (U/ml)b
SLE-IIF, which is dependent on anti-dsDNA Abs (24) and thus
reveal the effect of the apoptotic U937 cells and other autoantibodies. As shown in Table V, the ability of different SLE sera to
induce IFN-␣ production in PBMC, when combined with apoptotic U937 cells, was clearly associated with the occurrence of RNP
Abs but not anti-SSA, anti-SSB, anti-Sm, or anti-CL Abs. As expected, the IFN-␣ production caused by the SLE serum alone correlated only to the presence of anti-dsDNA Abs.
FIGURE 2. The effect of the caspase inhibitor zVAD-fmk on the
ability of the combination of SLE IgG and apoptotic U937 cells to
induced IFN-␣ production in PBMC. Apoptosis was induced in U937
cells by UV, anti-Fas Abs, or etoposide in the absence (䊐) or presence
(o) of zVAD-fmk (see Table I and Figs. 1 and 2), using untreated cells
as control. Normal PBMC were then cultured with the combination of
treated or control U937 cells and SLE IgG. Levels of IFN-␣ (U/ml;
means ⫾ SD) were measured by immunoassay after 24 h. PBMC alone
denotes cultures without U937 cells. Representative results from one of
three experiments are shown.
UV Treatment
Normal IgG
SLE IgGc
MonoMac6
MonoMac6
H9
H9
Jurkat
Jurkat
U266
U266
None
–
⫹
–
⫹
–
⫹
–
⫹
2⫾0
2⫾0
9⫾2
2⫾0
2⫾0
2⫾0
2⫾0
2⫾0
2⫾0
102 ⫾ 4
1058 ⫾ 191
292 ⫾ 41
276 ⫾ 50
109 ⫾ 44
494 ⫾ 64
87 ⫾ 5
191 ⫾ 89
13 ⫾ 11
a
Untreated or UV-treated (60 mJ) MonoMac6, H9, Jurkat, or U266 cells were first
precultured for 4 h and then cultured for 24 h with IFN-␣2b (500 U/ml)-costimulated
normal PBMC. The cultures contained either normal IgG or SLE IgG (1 mg/ml).
b
The figures represent IFN-␣ levels in culture medium (U/ml; means ⫾ SD)
measured by immunoassay.
c
IgG derived from a 66-year-old male patient with a SLEDAI score of 14.
Discussion
Apoptosis is usually considered to be a controlled process to eliminate damaged cells without causing an inflammatory response. In
fact, apoptotic cells have been shown to induce production of the
anti-inflammatory cytokines IL-10 and TGF-␤1 that can decrease
production of proinflammatory cytokines such as TNF-␣, IL-1␤,
and IL-12 (36, 37). Our finding that the combination of apoptotic
U937 cells and IgG from SLE patients can induce IFN-␣ production in human PBMC is therefore of considerable interest, because
IFN-␣ is a proinflammatory cytokine that can promote autoimmunity (9 –12, 38).
In our study, only the combination of SLE-IgG and apoptotic
U937 cells, but not either component alone, had IFN-␣-inducing
activity. A likely reason for the effect of SLE-IgG is the presence
of autoantibodies. A role for anti-DNA Abs were first considered,
because we previously demonstrated an endogenous circulating
IFN-␣ inducer in SLE patients consisting of anti-DNA Abs and
DNA as essential components (13, 24). Surprisingly, when IgG
FIGURE 3. IFN-␣2b increases the IFN-␣ production by normal PBMC
induced by the combination of SLE-IgG and apoptotic U937 cells. The
PBMC were cultured with different concentrations of recombinant IFN␣2b and the combination of SLE-IgG and either untreated U937 cells (䊐)
or UV-treated U937 cells (■). The levels of IFN-␣ (U/ml; mean ⫾ SD)
were measured after 24 h by immunoassay. Representative results from one
of four experiments.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
FIGURE 1. Anti-DNA Abs are not required for the ability of the combination of SLE-IgG and apoptotic U937 cells to cause IFN-␣ production
in normal PBMC. The IgG was purified from one SLE serum on a protein
G column and further separated on a dsDNA and/or a ssDNA cellulose
column. Effluents (see Materials and Methods) in combination with untreated (䊐) or UV-treated (■) U937 cells were added to PBMC cultures
and levels of IFN-␣ (U/ml; means ⫾ SD) at 24 h were determined by
immunoassay. Data from one of two experiments with similar results.
Cell Linea
The Journal of Immunology
3523
FIGURE 4.
The effect of the caspase inhibitor
zVAD-fmk on apoptosis induced in U937 cells. The
U937 cells were treated by UV light (60 mJ), anti-Fas
Abs (1 ␮g/ml) for 1 h, or etoposide (12 ␮M) for 3 h,
in the absence (o) or presence (䊐) of 50 ␮M zVADfmk. After 4 h of culture, apoptotic U937 cells were
detected by annexin V and PI staining (A) or by
TUNEL staining (B) as described in Materials and
Methods. Annexin V-positive, PI-negative cells were
considered apoptotic in A. The percentage of positive
cells was determined by flow cytometry, sample size
10,000 cells. Data from one of two experiments with
similar results.
ber of annexin V-positive and PI-negative cells did not decrease
among anti-Fas-treated U937 cells. This may indicate that zVADfmk failed to inhibit early apoptotic events in anti-Fas-treated
U937 cells, because annexin V binds to phosphatidylserine and
indicates an early phase of apoptosis (39). However, we observed
that the number of possibly necrotic annexin/PI double-positive
cells increased (results not shown). The latter may represent the
results of a Fas-mediated triggering of a necrotic pathway when
apoptosis is repressed, as has been reported in murine L929 cells
transfected with the human Fas gene (40). While our results indicate that cells dying by apoptosis can contribute to the induction of
IFN-␣ production, it is therefore not possible to exclude that other
forms of cell death may also be relevant.
We previously demonstrated that SLE-IIF in serum contains
DNA and anti-DNA Abs, and that such Abs from SLE patients
convert plasmid isDNA into a strong inducer of IFN-␣ synthesis in
normal PBMC (24). Furthermore, the presence of isDNA sequences in serum of SLE patients have been identified by molecular cloning and a pathogenic role suggested (41– 43), and similar
isDNA motifs consisting of unmethylated CpG palindromes have
been shown to induce IFN-␣ production (44 – 48). For these reasons, we speculate that isDNA could be generated by the U937
cells and be the actual IFN-␣ inducer in PBMC in the present
study. Such isDNA could be present as naked DNA, nucleosomes,
or larger chromatin fragments. It is relevant that nucleosomes can
be internalized in cells by Abs to histones or DNA, have biologic
Table IV. Induction of IFN-␣ production in PBMC by apoptotic U937 cells and IgG from different SLE patientsa
Induced IFN-␣b
Treatment of U937 cellsf
Culture
Supplementc
SLE-IgG
SLE-IgG
SLE-IgG
SLE-IgG
SLE-IgG
SLE-IgG
SLE-IgG
SLE-IgG
SLE-IgG
N-IgG 1
N-IgG 2
N-IgG 3
N-IgG 4
a
1
2
3
4
5
6
7
8
9
SLEDAI
Scored
10
5
4
22
0
0
4
0
0
dsDNA Abe
None
UV
Controls
112
239
2326
35
7
9
12
10
11
7
5
3
5
86 ⫾ 40
173 ⫾ 66
316 ⫾ 30
47 ⫾ 28
34 ⫾ 26
29 ⫾ 5
99 ⫾ 32
4⫾5
32 ⫾ 18
6⫾4
4⫾4
8⫾2
7⫾6
1393 ⫾ 389
1670 ⫾ 219
537 ⫾ 52
803 ⫾ 103
45 ⫾ 16
90 ⫾ 15
1012 ⫾ 166
16 ⫾ 5
93 ⫾ 22
11 ⫾ 9
⬍2 ⫾ 1
4⫾2
10 ⫾ 10
30 ⫾ 15
142 ⫾ 80
64 ⫾ 23
22 ⫾ 15
27 ⫾ 11
10 ⫾ 9
54 ⫾ 24
4⫾2
18 ⫾ 6
⬍2 ⫾ 0
2⫾1
⬍2 ⫾ 0
⬍2 ⫾ 0
Normal PBMC were cultured with the combination of U937 cells and IgG from SLE patients (SLE-IgG) and normal individuals (N-IgG) for 24 h.
The figures represent produced IFN-␣ (U/ml; mean ⫾ SD), measured by immunoassay. Results from one experiment.
IgG was prepared from sera by means of protein G columns and used in a final concentration of 1 mg/ml in the cultures.
d
Clinical disease activity in individual patients was determined by the SLEDAI score.
e
Anti-dsDNA Ab concentration (IU/ml) determined by immunoassay of sera used for IgG preparation.
f
The U937 cells were untreated or UV-treated (60 mJ) and then cultured for 4 h before addition of PBMCs. Controls denote PBMC cultured without U937 cells.
b
c
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fractions from different SLE patients were compared, we were unable to connect the IFN-␣ production caused by apoptotic U937
cells and SLE-IgG to the occurrence of anti-DNA Abs. Furthermore, removal of anti-DNA Abs by absorption to DNA-cellulose
had no effect on the IFN-␣-inducing capacity. In contrast, there
was a clear association between presence of anti-RNP Abs in SLE
sera and ability of the sera to induce IFN-␣ production when combined with apoptotic U937 cells. No such association could be
detected for the other Abs investigated: anti-SSA, anti-SSB, antiCL, anti-Sm, and anti-dsDNA. Further studies are necessary to
clarify whether it is the anti-RNP Abs or coexisting Abs with other
specificities that are involved in the IFN-␣ production.
With regard to the role of the U937 cells in the IFN-␣ production, apoptosis was important because pretreatment of these cells
with the apoptosis inducers etoposide or UV light greatly increased
their IFN-␣-inducing capacity. Furthermore, this increase was inhibited by pretreatment of U937 cells with the broad-spectrum
caspase inhibitor zVAD-fmk. However, somewhat discrepant results were obtained with anti-Fas Ab-treated U937 cells, because
the zVAD-fmk only caused a partial reduction of their IFN-␣inducing capacity. The latter finding may be related to our observation that zVAD-fmk inhibited cell death in a different way in
anti-Fas-treated U937 cells compared to UV light or etoposidetreated cells. Thus, the zVAD-fmk treatment clearly inhibited the
DNA fragmentation process triggered by all three apoptosis inducers, as determined by the TUNEL method. Paradoxically, the num-
APOPTOTIC U937 CELLS COMBINED WITH SLE-IgG INDUCE IFN-␣
3524
Table V. The occurrence of different autoantibodies in SLE sera is related to the ability of SLE sera alone or combined with UV-treated U937 cells
to cause IFN-␣ production in normal PBMC
Autoantibody
Specificity
Present
RNP ⫹
–
SSA ⫹
–
SSB ⫹
–
Sm ⫹
–
DNA ⫹
–
CL ⫹
–
IFN-␣ Production Due to UV-Treated U937b
n
a
4
18
8
14
3
19
1
21
7
15
4
17
Concentration
761 ⫾ 185
211 ⫾ 494
411 ⫾ 371
255 ⫾ 565
292 ⫾ 442
314 ⫾ 519
927
282 ⫾ 492
522 ⫾ 756
213 ⫾ 311
26 ⫾ 114
277 ⫾ 343
c
IFN-␣ Production Due to SLE Serum Alone
p
d
0.011
0.207
0.774
0.134
0.597
0.283
Concentration
p
22 ⫾ 10
130 ⫾ 284
37 ⫾ 59
152 ⫾ 319
65 ⫾ 102
117 ⫾ 277
12
115 ⫾ 265
315 ⫾ 404
14 ⫾ 12
292 ⫾ 541
37 ⫾ 52
0.766
0.759
0.566
0.636
⬍0.001
0.502
a
No. of patients (sera) investigated, same material as in Table I.
The IFN-␣ production in PBMC cultures caused by SLE serum alone subtracted from that caused by the combination of SLE serum and UV-treated U937 cells.
Concentration of IFN-␣ in 24 h culture medium (U/ml; mean ⫾ SD), determined by immunoassay.
d
Values of p when comparing IFN-␣-inducing ability of sera with and without the specified Ab using the Mann-Whitney U test.
b
c
We also found that the IFN-␣ production by PBMC cocultured
with SLE-IgG and apoptotic cells was markedly enhanced by IFN␣2b. Such a costimulatory effect of type I IFN on the IFN-␣ response, termed priming, has previously been reported mainly for
viral inducers and is under certain conditions necessary for
IFN-␣␤ gene transcription (55, 56). Such prominent priming effects of type I IFN were also noted for the IFN-␣ production induced by SLE-IIF and by plasmid DNA/anti-DNA Abs (24). Accordingly, IFN-␣ production caused by especially viral infections
in patients with inactive SLE might prime natural IPC to respond
to complexes of IgG and material from apoptotic cells and therefore initiate more vigorous and sustained synthesis of IFN-␣,
which would increase disease activity. Such a mechanism could
explain cases of disease relapses in SLE patients reported during
viral infections (57–59).
The hallmarks of active SLE include increased levels of apoptotic cells (25, 26, 28), presence of autoantibodies against DNA
and nucleic acid-associated proteins and several other autoantigens
(1), and ongoing IFN-␣ production (2, 3, 5, 7, 8, 60, 61). Thus, all
components seen in our in vitro system are present in SLE patients.
Therefore, it is possible that the same mechanisms by which SLEIgG and apoptotic cells induce IFN-␣ production in vitro also operates in vivo. Considering the immunostimulatory actions of
IFN-␣ (14 –19) and its ability to promote autoimmunity in humans
(9 –12, 38), the results of the present investigation can be crucial
for understanding the etiology and pathogenesis of SLE. Therefore, it is obviously important to further define the identity and
action of the active component(s) in the IgG fraction and in the
apoptotic U937 cells.
Acknowledgements
We thank Anders Perers for excellent technical assistance, Lotta Karlnell
for collecting sera from SLE patients, Drs. Helena Vallin and Anders Johannisson for invaluable technical advice, Dr. Bo Nilsson for autoantibody
determinations, Dr. Karl-Erik Johansson for performing assays for mycoplasma, and Dr. Dan Grandér for kindly providing the H9 and U266 cell
lines.
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