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Regulation of the interferon-╨Ю┬▒ production induced by RNA-containing immune complexes in plasmacytoid dendritic cells.

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ARTHRITIS & RHEUMATISM
Vol. 60, No. 8, August 2009, pp 2418–2427
DOI 10.1002/art.24686
© 2009, American College of Rheumatology
Regulation of the Interferon-␣ Production Induced by
RNA-Containing Immune Complexes in
Plasmacytoid Dendritic Cells
Maija-Leena Eloranta,1 Tanja Lövgren,1 Doreen Finke,1 Linda Mathsson,1 Johan Rönnelid,1
Berthold Kastner,2 Gunnar V. Alm,3 and Lars Rönnblom1
Objective. Interferon-␣ (IFN␣) is produced in
several autoimmune diseases, including systemic lupus
erythematosus (SLE), and may be important in their
pathogenesis. We undertook this study to investigate
how IFN␣ production induced by RNA-containing immune complexes (ICs) in plasmacytoid dendritic cells
(PDCs) is regulated.
Methods. Normal PDCs purified from peripheral
blood mononuclear cells (PBMCs) were cocultivated
with other cell populations isolated from healthy individuals or SLE patients. IFN␣ production was induced
by RNA-containing ICs, which consisted of anti-RNP
autoantibodies and U1 small nuclear RNP particles,
and the effects of prostaglandin E2 (PGE2), reactive
oxygen species (ROS), or the cytokines IFN␣2b,
granulocyte–macrophage colony-stimulating factor
(GM-CSF), interleukin-10 (IL-10), or tumor necrosis
factor ␣ (TNF␣) were explored.
Results. Monocytes inhibited IFN␣ production by
PDCs in PBMC cultures, while natural killer (NK) cells
were stimulatory. The monocytes had little effect on
IFN␣ production by pure PDCs but inhibited its stimulation by NK cells. Monocytes from SLE patients were
less inhibitory. Exposure of PBMCs or PDCs to
IFN␣2b/GM-CSF increased their IFN␣ production.
RNA-containing ICs caused production of ROS, PGE2,
and TNF␣, especially in monocytes. These mediators
and IL-10 suppressed IFN␣ production in PBMC cultures, with ROS and PGE2 also inhibiting IFN␣ production by purified PDCs. Inhibition by all of these
agents, except for ROS, was abolished by IFN␣2b/GMCSF. The inhibitory effect of monocytes was significantly counteracted by the ROS scavengers serotonin
and catalase.
Conclusion. IFN␣ production induced by RNAcontaining ICs in PDCs is regulated by a network of
interactions between monocytes, NK cells, and PDCs,
involving several pro- and antiinflammatory molecules.
This should be considered when designing and applying
new therapies.
Supported in part by grants from the Dana Foundation, the
Swedish Research Council, the Swedish Society of Medicine, the
Swedish Rheumatism Foundation, the Uppsala University Hospital
Development Foundation, the Agnes and Mac Rudberg Foundation,
the Swedish Fund for Research Without Animal Experiments, the
Nilsson Foundation, King Gustaf V’s 80-Year Foundation, and COMBINE.
1
Maija-Leena Eloranta, PhD, Tanja Lövgren, PhD, Doreen
Finke, PhD, Linda Mathsson, PhD, Johan Rönnelid, MD, PhD, Lars
Rönnblom, MD, PhD: Uppsala University, Uppsala, Sweden;
2
Berthold Kastner, PhD: Max Planck Institute of Biophysical Chemistry, Göttingen, Germany; 3Gunnar V. Alm, MD, PhD: Swedish
University of Agricultural Sciences, Uppsala, Sweden.
Address correspondence and reprint requests to Maija-Leena
Eloranta, PhD, Department of Medical Sciences, Clinical Research
Department 3, Systemic Autoimmunity Group, Entrance 85, 3rd
Floor, Uppsala University Hospital, S-75185 Uppsala, Sweden. E-mail:
maija-leena.eloranta@medsci.uu.se.
Submitted for publication November 20, 2008; accepted in
revised form April 18, 2009.
Systemic lupus erythematosus (SLE) is characterized by the occurrence of autoantibodies against nucleic
acids and associated proteins, formation of immune
complexes (ICs), and inflammation in many organs and
tissues. Many SLE patients have a continuous production of type I interferon (IFN), which is detected as
increased levels of IFN␣ and expression of type I
IFN–inducible genes (1–3). Type I IFNs have many
important immunoregulatory actions that could initiate
an autoimmune process (1,4), which is further promoted
by IFN␣-induced increase in autoantigen expression
(5,6). A pivotal role of type I IFN in the etiology and
pathogenesis of SLE has therefore been suggested and is
supported by many further observations (1,2). Thus,
IFN␣ levels and type I IFN–inducible gene expression
2418
REGULATION OF TYPE I IFN PRODUCTION
correlate with disease activity and severity as well as with
the levels of autoantibodies against double-stranded
DNA (dsDNA) and RNA binding proteins (7,8). In
addition, single-nucleotide polymorphisms in several
genes encoding key molecules involved in IFN production and action are strongly associated with SLE (9–11).
Furthermore, IFN␣ therapy in patients with malignancies or infections frequently results in autoimmune
adverse effects, including SLE-like disease (12,13). Finally, administration of antibodies neutralizing IFN␣ in
a phase I trial displayed promising efficacy in SLE
patients, indicating that IFN␣ is indeed involved in
pathogenesis and is a promising therapeutic target (14).
In SLE and several other systemic autoimmune
diseases, DNA– and RNA–protein complexes released
by apoptotic or necrotic cells form ICs with autoantibodies, and these ICs may serve as endogenous IFN
inducers (1,15–18). They activate type I IFN production
in plasmacytoid dendritic cells (PDCs) after endocytosis
mediated by Fc␥ receptor IIa (Fc␥RIIa) and activation
of endosomal Toll-like receptors (TLRs), TLR-7 by
single-stranded RNA and TLR-9 by unmethylated CpG
DNA (19–23).
Because of the important role of type I IFN in
SLE, it is essential to clarify the mechanisms that
regulate IFN␣ production induced by endogenous IFN
inducers such as RNA-containing ICs. We observed
previously that IFN␣ production triggered in normal
peripheral blood mononuclear cells (PBMCs) in vitro
by RNA-containing ICs was enhanced in the presence of
several different cytokines, such as IFN ␣ / ␤ and
granulocyte–macrophage colony-stimulating factor
(GM-CSF) (15). This phenomenon is termed priming
and is due to the induction of the transcription factors
IFN regulatory factor 5 (IRF-5) and IRF-7, which are
involved in the triggering of type I IFN genes (23). In
contrast, the cytokines interleukin-10 (IL-10) and tumor
necrosis factor ␣ (TNF␣) decreased this IC-induced
IFN␣ production (15). In the present study, we used
well-defined RNA-containing ICs consisting of purified
U1 small nuclear RNP (snRNP) and anti-RNP autoantibodies (21,22), and we observed initially that they were
poor inducers of IFN␣ production by the PDCs present
in PBMC cultures but strong inducers in highly purified
PDCs. These observations suggested a pronounced regulation of IFN␣ production by PDCs, which could be
important in the pathogenesis of systemic autoimmune
diseases.
In the present study, we therefore investigated in
more detail the regulatory effects of cells and their
mediators, including cytokines, on IFN␣ production
2419
induced by RNA-containing ICs. We found that monocytes from healthy individuals were potent inhibitors and
that natural killer (NK) cells were potent enhancers of
IFN␣ production in PBMC cultures. Because PDCs are
the actual IFN␣ producers, we investigated the extent to
which PDCs and NK cells were the target cells for the
suppressive monocytes and their potential mediators,
specifically reactive oxygen species (ROS), the prostanoid prostaglandin E2 (PGE2), and the cytokines TNF␣
and IL-10. We also studied the stimulatory (“priming”)
effects of cytokines IFN␣2b and GM-CSF on RNAcontaining IC–induced IFN␣ production in PDCs, as
well as how such priming especially affected monocytemediated inhibition.
PATIENTS AND METHODS
Patients and controls. Six SLE patients attending the
outpatient clinic at the Rheumatology Unit of Uppsala University Hospital were included in the study. All patients
fulfilled at least 4 (mean 5; range 4–6) of the American College
of Rheumatology criteria for SLE (24). The mean ⫾ SD age of
the patients was 40 ⫾ 11 years, and their mean ⫾ SD disease
duration was 18 ⫾ 12 years. All patients had low disease
activity (score of ⬍4 on the modified SLE Disease Activity
Index [25]). Healthy blood donors served as controls. The
study was approved by the local ethics committee, and informed consent was obtained from all patients and controls.
IFN inducers. U1 snRNP particles were purified from
HeLa cells as described previously (26,27). The U1 snRNP
preparations had a purity of at least 90%. Sera from a healthy
blood donor or an SLE patient containing autoantibodies to
SmB, SmD, U1 RNP A, and U1 RNP C proteins, ribosomal P
antigen, histone, and dsDNA were used to prepare normal IgG
and SLE IgG, respectively, by protein G chromatography. The
U1 snRNP particles were used at a final concentration of 2.5
␮g/ml in the cell cultures, together with a final concentration of
1 mg/ml of normal IgG or SLE IgG. The normal IgG was used
either untreated or aggregated by heating at 63°C for 60 minutes at a concentration of 50 mg/ml. Oligodeoxynucleotide
(ODN) 2216 (3 ␮g/ml; CyberGene, Huddinge, Sweden) was
used as TLR-9 agonist. Herpes simplex virus type 1 (HSV-1),
propagated in WISH cells and inactivated by ultraviolet light
(17), was used as a control IFN␣ inducer.
Cytokines, PGE2, and ROS. When indicated, the cultures were supplemented with IFN␣2b (500 units/ml, IntronA;
Schering-Plough, Bloomfield, NJ), GM-CSF (2 ng/ml, Leukine; Berlex, Montville, NJ), IL-10 (10 ng/ml; Genzyme,
Cambridge, MA), TNF␣ (100 units/ml; Genzyme), or PGE2
(100 ng/ml; Sigma-Aldrich, St. Louis, MO). H2O2 (Merck,
Darmstadt, Germany) was used at indicated concentrations.
ROS inhibitors. Serotonin hydrochloride, bovine liver
catalase, and human erythrocyte superoxide dismutase (SOD)
(all from Sigma-Aldrich) were used at final optimal concentrations of 200 ␮M, 100 units/ml, and 100 units/ml, respectively,
in the cultures.
2420
Cell preparation and culture conditions. PBMCs were
prepared by Ficoll-Hypaque (GE Healthcare, Uppsala, Sweden) density-gradient centrifugation of buffy coats from
healthy blood donors (Department of Transfusion Medicine,
Uppsala University Hospital) or of whole blood from SLE
patients and corresponding healthy controls. Pure cell populations were prepared from PBMCs by using the MACS magnetic labeling system (Miltenyi Biotec, Bergisch Gladbach,
Germany). Briefly, PDCs were purified (ⱖ95% purity) by
negative depletion using a PDC Isolation kit and MACS LS
columns (both from Miltenyi Biotec). CD56⫹ NK cells were
isolated using an NK Cell Isolation kit (Miltenyi Biotec)
(ⱖ96% purity), and CD14⫹ monocytes were prepared using
CD14 Microbeads (Miltenyi Biotec) (ⱖ97% purity). The purity of the isolated cell populations was determined using a
FACSCalibur flow cytometer (BD Biosciences, San Jose, CA)
after staining with fluorescein isothiocyanate–labeled anti–
blood dendritic cell antigen 2 (anti–BDCA-2; Miltenyi Biotec),
anti-CD56 (Miltenyi Biotec), or anti-CD14 (Serotec, Oxford,
UK) monoclonal antibodies (mAb).
Cells were cultured in 0.1-ml volumes in 96-well flatbottomed plates (Nunclon; Nunc, Roskilde, Denmark) using
Macrophage-SFM medium (Invitrogen, San Diego, CA) supplemented with HEPES (20 mM), penicillin (60 ␮g/ml), and
streptomycin (100 ␮g/ml). Cell concentrations were 2 ⫻ 106/ml
for PBMCs and 0.25 ⫻ 106/ml or 0.5 ⫻ 106/ml for isolated
PDCs, while monocytes and NK cells were cultured at 0.5–2 ⫻
106/ml as indicated. All cell cultures were set up as duplicates
in each experiment.
Immunoassays and ROS measurement. The IFN␣
concentrations in 20-hour culture supernatants were determined by a dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) as previously described (28). The anti-IFN␣
mAb LT27:293, which recognizes most IFN␣ subtypes except
IFN␣2b, was used as capture antibody, and the europiumlabeled anti-IFN␣ mAb LT27:297 was used as detection antibody. The IFN␣ standard was calibrated against the National
Institutes of Health reference leukocyte IFN␣ GA-23-02-530.
This assay has a detection limit of 2 units/ml. No IFN␣ production was detected in cell cultures without RNA-containing
ICs or HSV-1.
Concentrations of TNF␣ and IL-10 were determined
by enzyme-linked immunosorbent assay as previously described (29). Briefly, mAb to TNF␣ (MAB610; R&D Systems,
Minneapolis, MN) or IL-10 (9D7; PharMingen, San Diego,
CA) were used as capture antibodies, and biotin-conjugated
polyclonal antibodies to TNF␣ (BAF210; R&D Systems) or
biotin-conjugated mAb to IL-10 (12G8; PharMingen) were
used as secondary antibodies. Horseradish peroxidase–
conjugated streptavidin (R&D Systems) was used for detection. The lower detection limit is 2 pg/ml for IL-10 and 1 pg/ml
for TNF␣.
The levels of PGE 2 were determined using a
Prostaglandin EIA kit (Sigma-Aldrich) according to the manufacturer’s instructions. The lower detection limit for this assay
is 39 pg/ml.
ROS were assayed by a luminol-enhanced chemiluminescence system. The chemiluminescence activity was measured in a 6-channel Biolumat LB 9505 (Berthold, Wildbad,
Germany) using 105 PBMCs, CD14-depleted PBMCs, PDCs,
or monocytes in 0.1 ml of culture medium. Chemiluminescence
was measured continuously for 35 minutes at 37°C, beginning
ELORANTA ET AL
immediately after addition of luminol (5 mg/ml) and RNAcontaining ICs.
Statistical analysis. The significance of differences
between groups was calculated by t-test using GraphPad Prism
4.0 software (GraphPad Software, San Diego, CA). P values
less than or equal to 0.05 were considered significant.
RESULTS
IFN␣ production by PDCs in PBMC cultures
stimulated with RNA-containing ICs is deficient but is
enhanced by IFN␣2b/GM-CSF. We initially evaluated
the effect of IFN␣2b/GM-CSF priming on IFN␣ production by PBMCs or purified PDCs from healthy
individuals after stimulation with RNA-containing ICs,
consisting of U1 snRNP particles and anti-RNP–
containing IgG. For comparison, we used HSV-1, which,
like RNA-containing ICs, selectively induces IFN␣ production in PDCs (22). In addition to measuring levels of
IFN␣ (in units/ml) in the cultures, we calculated the
mean quantity of IFN␣ produced per PDC (units/PDC)
by dividing the total amount of IFN␣ in the cultures
(determined by the DELFIA) by the number of BDCA2–positive PDCs (determined by flow cytometry).
Total PBMCs stimulated with RNA-containing
ICs (Figure 1A) produced only very low amounts of
IFN␣ (corresponding to a mean of 0.01 units/PDC), and
these levels were increased by IFN␣2b/GM-CSF priming
(to a level corresponding to a mean of 0.05 units/PDC)
(P ⫽ 0.006) (Table 1). In contrast, IFN␣ production
induced by HSV-1 in PBMCs was considerably higher
and was not increased by priming (Figure 1A and Table
1). Compared with PBMCs, purified PDCs stimulated
with RNA-containing ICs or HSV-1 produced much
higher levels of IFN␣ (Figure 1A), corresponding to 0.09
units/PDC (Table 1). Priming the purified PDCs with
IFN␣2b/GM-CSF significantly increased IFN␣ levels
induced by RNA-containing ICs (P ⫽ 0.006) and HSV-1
(P ⫽ 0.019) (Figure 1A), resulting in 0.28 units/PDC and
0.21 units/PDC, respectively (Table 1).
Thus, RNA-containing ICs and HSV-1 are both
potent IFN␣ inducers in purified PDCs, but RNAcontaining ICs are very poor inducers of IFN␣ production by PDCs in PBMC cultures. With both IFN inducers, IFN␣ production increased with IFN␣2b/GM-CSF
priming, strikingly so in the case of RNA-containing ICs.
Monocytes inhibit IFN␣ production induced by
RNA-containing ICs. The reason for the low IFN␣
production by PDCs in PBMCs from healthy individuals
stimulated with RNA-containing ICs could be partly
related to a lack of costimulation, since the impaired
IFN␣ response was increased by priming with IFN␣2b/
GM-CSF. Another explanation could be a suppressive
REGULATION OF TYPE I IFN PRODUCTION
2421
sponsible for the low IFN␣ production by PBMCs in
response to RNA-containing ICs.
Indeed, RNA-containing ICs induced higher levels of IFN␣ in CD14-depleted PBMCs compared with
whole PBMC cultures, both with (P ⫽ 0.032) and
without (P ⫽ 0.013) IFN␣2b/GM-CSF priming (Figure
1B). However, the HSV-1–induced IFN␣ production by
unprimed CD14-depleted PBMCs and intact PBMCs
did not differ (P ⫽ 0.09) and was only moderately
increased in CD14-depleted PBMCs compared with
intact PBMCs (P ⫽ 0.034) when primed with IFN␣2b/
GM-CSF (Figure 1C). Purified monocytes produced no
IFN␣ (⬍10 units/ml) when stimulated with either IFN
inducer.
We also investigated whether CD14⫹ monocytes
actually inhibited IFN␣ production by purified PDCs
stimulated with RNA-containing ICs (Figure 2A). However, addition of even high numbers of purified CD14⫹
monocytes had little or no inhibitory effect (P ⫽ 0.75).
In contrast, IFN␣ production by PDCs was markedly
increased in cocultures with CD14-depleted PBMCs
(P ⫽ 0.007; n ⫽ 6). This increase was prevented by the
addition of CD14⫹ monocytes to such cultures (Figure 2B).
Thus, monocytes had a profound negative impact
on IFN␣ production induced by RNA-containing ICs in
PBMC cultures. However, this was not a direct effect on
PDCs, the actual IFN␣ producers. Instead, the monocytes appeared to inhibit the enhancing effect of other
cells among the PBMCs on IFN␣ production by PDCs.
IFN␣ production by PDCs stimulated with RNAcontaining ICs is enhanced by CD56ⴙ NK cells. Because NK cells can promote CpG-induced PDC activaFigure 1. Decreased interferon-␣ (IFN␣) production by plasmacytoid
dendritic cells (PDCs) in cultures of peripheral blood mononuclear cells
(PBMCs) stimulated with RNA-containing immune complexes (RNAIC), consisting of U1 small nuclear RNP particles and anti-RNP–
containing IgG, is associated with the presence of monocytes and is
increased by priming with IFN␣2b/granulocyte–macrophage colonystimulating factor (GM-CSF). A, IFN␣ production in cultures of
PBMCs or purified PDCs (0.5 ⫻ 105 per well) stimulated with
RNA-containing ICs or herpes simplex virus type 1 (HSV-1), with or
without priming with IFN␣2b/GM-CSF. B and C, IFN␣ production by
PBMCs, monocyte-depleted PBMCs (CD14⫺), or purified monocytes
(CD14⫹). The cells (0.2 ⫻ 106 per culture) were stimulated with
RNA-containing ICs (B) or HSV-1 (C), with or without IFN␣2b/GMCSF priming. The levels of IFN␣ in cell culture medium were
determined by immunoassay after 20 hours. Horizontal lines show the
median of 5 experiments. NS ⫽ not significant.
action of other PBMC types on the PDCs. We therefore
investigated whether the CD14⫹ monocytes were re-
Table 1. Quantity of IFN␣ produced per PDC in cultures of PBMCs
or purified PDCs stimulated with RNA-containing ICs or HSV-1*
IFN␣ produced per PDC, units
Inducer, cell
population
RNA-containing ICs
PBMCs
PDCs
HSV-1
PBMCs
PDCs
No
priming
IFN␣2b/GM-CSF
priming
P†
0.01 ⫾ 0.006
0.09 ⫾ 0.08
0.05 ⫾ 0.02
0.28 ⫾ 0.14
0.006
0.006
0.16 ⫾ 0.060
0.09 ⫾ 0.04
0.19 ⫾ 0.16
0.21 ⫾ 0.11
NS
0.019
* Values are the mean ⫾ SD of 5 experiments. IFN␣ ⫽ interferon-␣;
PDC ⫽ plasmacytoid dendritic cell; PBMCs ⫽ peripheral blood
mononuclear cells; RNA-containing ICs ⫽ immune complexes consisting of U1 small nuclear RNP particles and anti-RNP–containing IgG;
HSV-1 ⫽ herpes simplex virus type 1; GM-CSF ⫽ granulocyte–
macrophage colony-stimulating factor; NS ⫽ not significant.
† By paired t-test. P values less than or equal to 0.05 were considered
significant.
2422
ELORANTA ET AL
Figure 2. Effect of monocytes, monocyte-depleted PBMCs, or natural killer (NK) cells on IFN␣
production by PDCs stimulated with RNA-containing ICs (A–C) or oligodeoxynucleotide (ODN) 2216 (D).
A, IFN␣ production by PDCs alone (0.25 ⫻ 105 per well), CD14-depleted PBMCs (CD14–) alone, or PDCs
together with indicated numbers of either purified monocytes (CD14⫹) or CD14-depleted PBMCs. B,
IFN␣ production by PDCs alone (0.25 ⫻ 105 per well) or by PDCs cocultivated either with CD14-depleted
PBMCs or with CD56⫹ NK cells (CD56⫹) in the absence or presence of CD14⫹ monocytes. C, IFN␣
production by CD56⫹ NK cells alone or by PDCs alone (0.25 ⫻ 105 per well) or cocultivated with indicated
numbers of either CD14⫺CD56⫺ PBMCs or CD56⫹ NK cells. D, ODN2216-triggered IFN␣ production
by PDCs alone (0.25 ⫻ 105 per well), cocultivated with CD56⫹ NK cells, cocultivated with CD56⫹ NK cells
and CD14⫹ monocytes in the absence or presence of heat-aggregated normal IgG. Values are the mean.
Results are from 1 representative experiment of 3 performed. See Figure 1 for other definitions.
tion, we investigated whether NK cells were responsible
for the observed ability of CD14-depleted PBMCs to
enhance RNA-containing IC–induced IFN␣ production
by PDCs (30). Although the purified PDCs produced
variable and often low levels of IFN␣ in response to
RNA-containing ICs, their IFN␣ production was dramatically increased (P ⫽ 0.004) by cocultivation with
purified NK cells (Figures 2B and C). Similar effects of
NK cells were seen using HSV-1 as IFN␣ inducer
(results not shown). In contrast, only small stimulatory
effects were seen with NK-depleted CD14⫺ PBMCs
(Figure 2C). The purified CD56⫹ NK cells alone produced no IFN␣ when stimulated with RNA-containing
ICs (Figure 2C).
We also investigated whether autologous CD14⫹
monocytes from healthy individuals inhibited the stimulatory effect of NK cells. We found that monocytes
strongly reduced RNA-containing IC–induced IFN␣
production when added to cocultures of PDCs with
CD56⫹ NK cells (Figure 2B). Consequently, NK cells
can enhance RNA-containing IC–induced IFN␣ production by PDCs, but this effect is inhibited by monocytes.
Next, we investigated whether the inhibitory effect of
monocytes was due to TLR ligation or required Fc␥R
activation (Figure 2D). Cultures of PDCs, CD56⫹ NK
cells, and monocytes from healthy blood donors were
stimulated with the TLR-9 agonist ODN2216, and heataggregated normal IgG was added to the cells. We found
that IFN␣ production was down-regulated only in the
Figure 3. Less suppression of IFN␣ production by monocytes from
patients with systemic lupus erythematosus (SLE). PDCs and CD56⫹
natural killer cells were cocultivated with CD14⫹ monocytes isolated
from SLE patients (n ⫽ 6) and healthy blood donors (n ⫽ 5), and
IFN␣ levels in supernatants were determined by immunoassay after 20
hours of culture in all experiments. Horizontal lines show the median.
See Figure 1 for other definitions.
REGULATION OF TYPE I IFN PRODUCTION
2423
presence of heat-aggregated normal IgG. This indicated
that a signal through Fc␥R was required for triggering
the inhibitory effect of monocytes.
We also compared the ability of monocytes from
SLE patients and healthy controls to inhibit NK cell–
enhanced IFN␣ production in PDCs induced by RNAcontaining ICs. Monocytes from SLE patients appeared
less suppressive (Figure 3).
Suppression of RNA-containing IC–induced
IFN␣ production by cytokines, PGE2, and ROS. Cytokines or other inflammatory mediators could be responsible for the suppressive effect of monocytes on RNAcontaining IC–induced IFN␣ production. We therefore
measured the levels of TNF␣, IL-10, PGE2, and ROS in
Figure 4. Tumor necrosis factor ␣ (TNF␣), prostaglandin E2 (PGE2),
and reactive oxygen species (ROS) production induced by RNAcontaining ICs. A, PGE2 or TNF␣ production by 0.2 ⫻ 106 PBMCs,
CD14-depleted PBMCs (CD14⫺), or purified CD14⫹ monocytes
(CD14⫹) stimulated with RNA-containing ICs or medium only
(Mock). The levels of PGE2 or TNF␣ in 20-hour culture medium were
determined by immunoassay. B, ROS production by 0.1 ⫻ 106 PBMCs,
PDCs, CD14-depleted PBMCs, or purified CD14⫹ monocytes. ROS
production was measured with a luminol-enhanced chemiluminescence method for 30 minutes beginning immediately after addition of
RNA-containing ICs. Values are the mean. Results are from 1
representative experiment of 3 performed. See Figure 1 for other
definitions.
Figure 5. Inhibitory effects of prostaglandin E2 (PGE2), interleukin-10
(IL-10), tumor necrosis factor ␣ (TNF␣), and reactive oxygen species
(H2O2) on IFN␣ production induced by RNA-containing ICs. A and B,
IFN␣ production by PBMCs (A) or PDCs (B), with or without
IFN␣2b/GM-CSF priming, stimulated with RNA-containing ICs in the
presence of the indicated concentrations of PGE2, IL-10, or TNF␣. C,
Effect of H2O2 on PDCs stimulated with RNA-containing ICs. IFN␣
levels in supernatants were determined by immunoassay after 20 hours
of culture. Values are the mean. Results are from 1 of 3 experiments,
each of which showed similar results. See Figure 1 for other definitions.
the cell cultures and found that RNA-containing ICs
induced production of PGE2 and TNF␣ (Figure 4A) as
well as ROS (Figure 4B) by PBMCs and purified
monocytes from healthy individuals. Monocyte-depleted
PBMCs produced TNF␣ but little or no PGE2 and ROS
(Figure 4), as did purified PDCs (results not shown for
TNF␣ and PGE2). No ROS production was detected in
PBMCs, PDCs, monocyte-depleted PBMCs, or monocytes without RNA-containing ICs (not shown). The
2424
ELORANTA ET AL
Figure 6. Effects of reactive oxygen species scavengers on IFN␣ production induced by RNA-containing ICs. PBMCs and
CD14-depleted PBMCs were stimulated with RNA-containing ICs in the presence of 200 ␮M serotonin or 100 units/ml
catalase (A) and 100 units/ml superoxide dismutase (SOD) (B), using medium only as control. The levels of IFN␣ after
20 hours of culture were determined by immunoassay. Values are the mean and SD. Results are from 1 of 3 experiments,
each of which showed similar results. See Figure 1 for other definitions.
levels of IL-10 were low in all cultures (⬍20 pg/ml). In
general, similar levels of mediators were seen when cells
were primed with IFN␣2b/GM-CSF (results not shown).
The RNA-containing IC is therefore a potent activator
of monocytes, inducing production of at least PGE2,
TNF␣, and ROS.
We further investigated whether PGE2 and the
cytokines IL-10 and TNF␣ actually affected IFN␣ production by PDCs. In unprimed PBMC cultures, all these
mediators were found to inhibit RNA-containing IC–
induced IFN␣ production, but no inhibition was seen
with IFN␣2b/GM-CSF–primed cells (Figure 5A). In
cultures of purified PDCs, PGE2 caused a strong inhibition (88%) of IFN␣ production induced by RNAcontaining ICs, but not in the presence of IFN␣2b/
GM-CSF priming (Figure 5B). In contrast, TNF␣ and
IL-10 had no significant inhibitory effects on the purified
PDCs.
With regard to the effect of ROS administered as
H2O2, higher concentrations had an inhibitory effect on
both purified PDCs (Figure 5C) and PBMCs (results not
shown), regardless of IFN␣2b/GM-CSF priming. Because it is difficult to mimic the different ROS in vitro,
we determined whether blocking of ROS production
with the scavengers serotonin, catalase (Figure 6A), or
SOD (Figure 6B) could restore IFN␣ production in
PBMC cultures stimulated with RNA-containing ICs.
All 3 agents were found to increase IFN␣ production by
PBMCs, but none of them had any effect on CD14depleted PBMCs. This suggests that monocytes were
responsible for ROS production and down-regulation of
the type I IFN response.
DISCUSSION
In the present study, we examined the regulation
of IFN␣ production induced in normal PDCs by RNAcontaining ICs consisting of U1 snRNP and SLE IgG,
which are representative of the endogenous IFN inducers that are considered to be responsible for the activation of type I IFN production in several systemic
autoimmune diseases (1,2). We initially observed exceptionally low IFN␣ production induced by RNAcontaining ICs in cultures of PBMCs from healthy
individuals, while RNA-containing ICs and HSV-1 were
both potent stimulators of purified PDCs. Costimulation
(priming) with IFN␣2b/GM-CSF increased the amount
of IFN␣ produced by both PBMCs and PDCs. However,
in the case of PBMCs stimulated with RNA-containing
ICs, the amount of IFN␣ produced per PDC in the
PBMC cultures was still far below estimates of the
IFN␣-producing capacity of PDCs in previous studies
(23) and also for purified PDCs in the present study.
The specific removal of the CD14⫹ monocytes
from PBMCs restored the IFN␣ production stimulated
with RNA-containing ICs to the same level as that
stimulated with HSV-1. Furthermore, addition of monocytes to monocyte-depleted PBMCs inhibited their
RNA-containing IC–induced IFN␣ production. Consequently, monocytes suppressed IFN␣ production of PBMCs in response to RNA-containing ICs. Unexpectedly,
they had little effect on the purified PDCs. Because we
observed that monocyte-depleted PBMCs actually
strongly increased IFN␣ production by purified PDCs,
we explored the possibility that the monocytes actually
REGULATION OF TYPE I IFN PRODUCTION
were suppressive of one or several types of PBMCs that
enhanced IFN␣ production by PDCs.
Indeed, purified NK (CD56⫹) cells from healthy
individuals were found to be highly efficient enhancers
of IFN␣ production by PDCs upon stimulation with
either RNA-containing ICs or HSV-1. Conversely, elimination of NK cells essentially abrogated the enhancing
effects of the monocyte-depleted PBMCs. This NK
stimulation was especially pronounced at low PDC responses, with ⬃20-fold increases in the IFN␣ levels.
Consequently, NK cell function is important in determining the PDC response to physiologic IFN inducers
(i.e., RNA-containing ICs and HSV-1), and our results
are consistent with findings that NK cells increase IFN␣
production by PDCs stimulated with synthetic unmethylated CpG-containing ODNs (30,31). Because PDCs
and NK cells coexist in tissues, including secondary
lymphoid organs (32), NK cells could clearly be a major
physiologic regulator of IFN␣ production by PDCs,
which is possibly also relevant in the activation of type I
IFN production in SLE patients. However, the mechanism of the enhancing action of NK cells needs to be
further investigated, including the molecular basis of
PDC–NK cell interactions.
The observation that CD14⫹ monocytes strongly
inhibited the NK cell–mediated enhancement of IFN␣
production by PDCs induced by RNA-containing ICs,
via interaction with Fc␥R, raised the question of
whether RNA-containing ICs could activate the production of inflammatory mediators in monocytes, as described for other ICs (29,33,34). Such mediators include
cytokines, prostanoids, and ROS, which could suppress
IFN␣ production by direct inhibitory effects on PDCs or
on stimulatory NK cells. In addition, ROS could reduce
the proinflammatory properties of high mobility group
box chromosomal protein 1 (35). Indeed, we demonstrated that RNA-containing ICs caused production of
ROS, TNF␣, and PGE2, but not IL-10, in monocytes
from healthy individuals. However, the latter cytokine
has been reported to be produced by monocytes activated by other types of ICs (36). All 4 mediators we
examined (PGE2, TNF␣, IL-10, ROS) were found to
inhibit IFN␣ production by normal human PBMCs that
had been stimulated with RNA-containing ICs, but only
ROS (as H2O2) and PGE2 were inhibitory of purified
PDCs. Interestingly, all agents except H2O2 lost all or
most inhibitory activity when PBMCs or PDCs were
exposed to IFN␣2b/GM-CSF. Using the scavenging inhibitors catalase, serotonin, and SOD, we confirmed that
the inhibitory activity of monocytes was due to ROS to
a significant extent. However, because monocytes had
2425
little impact on IFN␣ production by purified PDCs in
cocultures, PDCs may also be relatively resistant to ROS
and other monocyte-derived mediators.
These new findings suggest that the primary
target cells for monocytes and their mediators are not
PDCs. Instead, NK cells are a particularly interesting
candidate target, because PGE2 (37,38), IL-10 (39), and
TNF␣ (40) have all been reported to impair NK cell
functions. Furthermore, NK cells are very sensitive to
ROS and are rendered apoptotic by ROS produced by
monocyte/macrophages or delivered as exogenous H2O2
(41,42). In addition, we noticed that monocytes from
SLE patients were less efficient inhibitors of IFN␣
production when added to cocultures with PDCs and
NK cells from healthy individuals. Monocytes may therefore be part of an important feedback mechanism that
limits the autoimmune reaction driven by IFN␣, and it is
conceivable that this function is deficient in SLE. This
conclusion is also supported by the observation that
monocytes from SLE patients have a deficient production of ROS (43) and suggests that normalization of the
monocyte/ROS system in SLE could ameliorate the type
I IFN–driven disease process.
The observation that IFN␣2b/GM-CSF increased
the otherwise poor ability of normal PBMCs to produce
IFN␣ upon stimulation with RNA-containing ICs is
consistent with previous data showing such priming by
IFN␣ on responses to several different IFN␣ inducers.
These IFN␣ inducers include interferogenic ICs formed
by apoptotic or necrotic cell material mixed with autoantibodies (16,17,44). The priming phenomenon is commonly thought to be due to direct effects of IFN␣ on
PDCs, resulting in increased expression of proteins
involved in type I IFN gene expression, including the
IFN-inducible transcription factors IRF-5 and IRF-7
(23). Increased survival of PDCs is another possibility,
because we noted that IFN␣2b/GM-CSF prevented
PGE2-mediated inhibition of the IFN␣ production in
purified PDCs, and PGE2 is known to cause apoptosis
and inhibition of IFN␣ production in these cells (45).
However, IFN␣2b/GM-CSF may also act on other cells
in the PBMC population that inhibit or stimulate PDC
functions (e.g., monocytes and NK cells). This possibility
is supported by our findings that IFN␣2b/GM-CSF
prevented the inhibitory effects of the cytokines IL-10
and TNF␣. Because SLE patients have signs of both
type I IFN (1,2) and GM-CSF (46) activation, the
enhancing effects of IFN␣2b/GM-CSF on IFN␣ production may be relevant in vivo by sustaining the disease
process.
We conclude that IFN␣ production induced by
2426
ELORANTA ET AL
RNA-containing ICs in PDCs is suppressed by monocytes and that mediators such as ROS, PGE2, and the
cytokines TNF␣ and IL-10 can be involved. However,
the production of IFN␣ by PDCs is enhanced by NK
cells and also by PDCs themselves via production of type
I IFN. Consequently, there is a network of interactions
between at least 3 types of cells that regulate IFN␣
production by PDCs, the details of which should be
further elucidated. This is important, considering the
crucial role of the type I IFN system in several systemic
autoimmune diseases, and may both increase our understanding of their pathogenesis and reveal new therapeutic targets.
ACKNOWLEDGMENTS
9.
10.
11.
12.
13.
14.
We thank Ms Anne Trönnberg for excellent technical
assistance and Ms Annika Hult for help with the ROS analysis.
We also want to thank Dr. Keith Elkon for helpful discussions.
15.
AUTHOR CONTRIBUTIONS
16.
All authors were involved in drafting the article or revising it
critically for important intellectual content, and all authors approved
the final version to be published. Dr. Eloranta had full access to all of
the data in the study and takes responsibility for the integrity of the
data and the accuracy of the data analysis.
Study conception and design. Eloranta, Lövgren, Alm, Rönnblom.
Acquisition of data. Eloranta, Lövgren, Finke, Mathsson, Rönnelid,
Kastner.
Analysis and interpretation of data. Eloranta, Lövgren, Finke, Rönnelid, Alm, Rönnblom.
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