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Expression of the markers BDCA-2 and BDCA-4 and production of interferon-╨Ю┬▒ by plasmacytoid dendritic cells in systemic lupus erythematosus.

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Vol. 48, No. 9, September 2003, pp 2524–2532
DOI 10.1002/art.11225
© 2003, American College of Rheumatology
Expression of the Markers BDCA-2 and BDCA-4 and
Production of Interferon-␣ by Plasmacytoid Dendritic Cells in
Systemic Lupus Erythematosus
Stina Blomberg,1 Maija-Leena Eloranta,2 Mattias Magnusson,2 Gunnar V. Alm,2
and Lars Rönnblom1
Objective. To study the expression of blood dendritic cell antigen 2 (BDCA-2) and BDCA-4 molecules
by plasmacytoid dendritic cells (PDCs) in the blood of
patients with systemic lupus erythematosus (SLE), and
to study PDC production of interferon-␣ (IFN␣) and its
inhibition by anti–BDCA-2 and anti–BDCA-4 antibodies.
Methods. Peripheral blood mononuclear cells
(PBMCs) from SLE patients (SLE PBMCs) and from
healthy controls were induced to produce IFN␣ in vitro
by SLE serum containing an endogenous IFN␣inducing factor (SLE-IIF) or by herpes simplex virus
type 1 (HSV-1). The frequencies and numbers of BDCA2–, BDCA-3–, and BDCA-4–expressing cells were analyzed by flow cytometry, and the effects of anti–BDCA-2
and anti–BDCA-4 monoclonal antibodies (mAb) on
IFN␣ production were investigated.
Results. IFN␣ production by SLE PBMCs induced by SLE-IIF or HSV-1 was decreased compared
with that of healthy control PBMCs (P ⴝ 0.002 and P ⴝ
0.0007, respectively). The proportions of BDCA-2– and
BDCA-3–expressing cells in SLE PBMCs were reduced
compared with those in PBMCs from healthy controls
(P ⴝ 0.01 and P ⴝ 0.004, respectively). IFN␣ producers
in culture, especially among SLE PBMCs, displayed
reduced BDCA-2 expression and constituted only a
minority of the BDCA-2–positive cells, at least in
healthy control PBMCs (median 18%). IFN␣ production by both SLE and healthy control PBMCs stimulated by SLE-IIF or HSV-1 was markedly reduced by
anti–BDCA-2 mAb (median 81–98% inhibition). Anti–
BDCA-4 mAb only partially inhibited SLE-IIF–induced
IFN␣ production.
Conclusion. SLE patients had a reduced number
of BDCA-2–expressing PDCs, also termed natural
IFN␣-producing cells, and their IFN␣ production could
be inhibited by anti–BDCA-2/4 mAb. Such mAb may be
a therapeutic option for inhibiting the ongoing IFN␣
production in SLE patients.
Patients with systemic lupus erythematosus
(SLE) have an ongoing production of interferon-␣
(IFN␣), and serum levels of IFN␣ correlate with both
disease activity and severity (1,2). Such IFN␣ may have
an important role in the pathogenesis of the disease,
because IFN␣ levels correlate with several markers of
immune activation considered to be of fundamental
importance in the disease process, such as anti–doublestranded DNA (anti-dsDNA) antibody levels, complement activation, and serum levels of interleukin-10
(IL-10) (2). More direct support for a causative role of
IFN␣ in the pathogenesis of SLE comes from the
observation that IFN␣ treatment of patients with nonautoimmune diseases may cause the development of
antinuclear antibodies, anti-dsDNA antibodies, and, occasionally, an SLE syndrome (3–8). Consequently, the
IFN␣ in SLE may break tolerance and initiate a de novo
autoimmune reaction, which eventually leads to autoimmune disease. This is perhaps not surprising considering the many immunoregulatory effects of IFN␣, such
as the promotion of survival and differentiation of
Supported in part by grants from the Swedish Research
Council, the Swedish Rheumatism Foundation, the Swedish Society of
Medicine, the 80-Year Foundation of King Gustaf V, the Åke Wiberg
Foundation, the Nanna Svartz Foundation, and the Magnus Bergvall
Stina Blomberg, MD, Lars Rönnblom, MD, PhD: Uppsala
University Hospital, Uppsala, Sweden; 2Maija-Leena Eloranta, PhD,
Mattias Magnusson, MsSci, Gunnar V. Alm, MD, PhD: Swedish
University of Agricultural Sciences, Uppsala, Sweden.
Address correspondence and reprint requests to Stina
Blomberg, MD, Immunology (V), Biomedical Center, Post Office Box
588, 751 23 Uppsala, Sweden. E-mail:
Submitted for publication November 11, 2002; accepted in
revised form May 9, 2003.
antigen-activated T and B lymphocytes (9–13), induction
of Fas ligand–mediated apoptosis (14), and maturation
of functionally active dendritic cells (DCs) (15–17).
The reason for the continuous IFN␣ production
in SLE has largely been unknown, but we recently
described the occurrence of a circulating endogenous
IFN␣ inducer in SLE patients (SLE-IIF), consisting of
immune complexes containing IgG and DNA (18,19).
These complexes are sometimes as potent in vitro as
viruses in inducing IFN␣ production in peripheral blood
mononuclear cells (PBMCs) from healthy blood donors,
and they selectively activate the natural IFN␣-producing
cells (NIPCs). The NIPCs are the most efficient IFN␣producing cells in vitro, with the capacity to synthesize
1–2 units of IFN␣ per cell (for review, see ref. 20), and
they were described earlier as immature DCs (21). They
share the phenotype of CD11c⫺,CD123⫹ DC precursors (22,23), which are now often referred to as plasmacytoid DCs (PDCs) and which have been verified to be
the principal IFN␣-producing cells (24,25). The PDCs
can function as antigen-presenting cells and produce
immunoregulatory cytokines such as IFN␣ and IL-12,
and in this way, they can link innate and adaptive
immunity (26,27).
SLE patients have reduced numbers of functionally normal NIPCs/PDCs in the circulation (18), but they
have increased numbers of PDCs in cutaneous lesions
(28) and active IFN␣-producing cells in both cutaneous
lesions and unaffected skin (29). Because the NIPCs/
PDCs are also the principal IFN␣ producers activated by
endogenous SLE-related IFN␣ inducers, such as SLEIIF or the combination of autoantibodies and apoptotic
cells (19,30), these cells may play a pivotal role in the
etiopathogenesis of SLE (31,32).
Two markers have recently been described that
identify the NIPC/PDC population, the blood dendritic
cell antigens (BDCAs) 2 and 4 (33). The BDCA-2
molecule is identified as a type II C-type lectin that may
be involved in ligand internalization, processing, and
presentation (33,34). Although the proportion of IFN␣
producers among BDCA-2/4–positive NIPCs/PDCs is
unknown, it is interesting that antibodies directed
against BDCA-2 can inhibit the IFN␣ production triggered by SLE-IIF or by viral inducers in PBMCs from
healthy blood donors (34). Such antibodies were suggested to be of potential therapeutic value in SLE, but
their efficacy on NIPCs/PDCs from SLE patients remains to be determined. In the present study, we
therefore investigated the NIPC/PDC population in SLE
patients using the BDCA-2 and BDCA-4 markers. We
also included a third marker, BDCA-3, which detects a
CD11c⫹,CD123⫺ subset of DCs (33). We investigated
whether the DC subsets identified by these 3 BDCA
markers were decreased in SLE patients. We also examined the IFN␣-producing capacity of the NIPC/PDC
population in SLE patients, studied whether the IFN␣producing capacity of PBMCs was related to the number
of PDCs expressing BDCA-2 and BDCA-4, and investigated whether BDCA-2/4 ligation affected the IFN␣producing capacity.
Patients and controls. Consecutive patients entering
the rheumatology clinic who fulfilled the American College of
Rheumatology 1982 revised classification criteria for SLE (35)
were selected for this investigation. Among the 35 patients, 2
were untreated and 6 received prednisolone as monotherapy
(mean dosage 8 mg/day, range 7.5–10 mg/day). The majority,
22 patients, were treated with both prednisolone (mean dosage
9.3 mg/day, range 5–60 mg/day) and a complementary therapy;
17 received chloroquine (160–300 mg/day), 9 received azathioprine (50 or 100 mg/day), and 1 patient each received methotrexate, colchicine, gamma globulin, or cyclosporine in addition to the steroid regimen. Two patients received chloroquine
as monotherapy, and 1 patient received chloroquine and
azathioprine without steroids. Disease activity was scored in all
SLE patients by a modified SLE Disease Activity Index
(SLEDAI) (36) excluding anti-dsDNA antibodies and complement activity. At the time of their inclusion in the study, 8
patients showed elevated SLEDAI scores (range 1–16) due to
arthritis, new skin rash, thrombocytopenia, or pathologic urinary sediment/glomerulonephritis. The 35 patients (32 women
and 3 men) had a median age of 42 years (range 19–67 years).
A total of 24 staff members (23 women and 1 man) with a
median age of 40 years (range 25–64 years) served as controls.
The study protocol was approved by the Committee of Ethics
of the Faculty of Medicine of Uppsala University.
Preparation of PBMCs. Blood samples obtained from
patients and healthy controls were collected into heparinized
tubes (Vacutainer; Becton Dickinson, Rutherford, NJ), and
PBMCs were prepared by Ficoll-Hypaque (Amersham Biosciences, Uppsala, Sweden) density gradient centrifugation.
Cells were washed 4 times in phosphate buffered saline (PBS)
and suspended in RPMI 1640 medium (Flow, Irvine, UK) with
penicillin (60 ␮g/ml), streptomycin (100 ␮g/ml), L-glutamine (2
mM), 20 mM HEPES, and 0.2 mg/ml human serum albumin
(HSA; Pharmacia, Stockholm, Sweden).
IFN␣ inducers. Herpes simplex virus type 1 (HSV-1)
was prepared as described previously (21) and used after being
ultraviolet irradiated (1J at 254 nm). A large plasma sample
was collected by plasmapheresis from a 16-year-old female
SLE patient with a SLEDAI score of 10. The citrated plasma
sample was converted to serum by addition of 1M CaCl2 and
stored at ⫺80°C.
IFN␣ induction cultures. PBMCs were stimulated by
HSV-1 or by 12.5% SLE serum in duplicate cultures in 96-well
flat-bottomed microtiter plates (Nunclon; Nunc, Roskilde,
Denmark) at a final concentration of 1 ⫻ 106/ml and final
volumes of 200 ␮l/well. Cultures were supplemented with 500
units/ml recombinant IFN␣ (Intron-A; Schering-Plough,
Bloomfield, NJ) and 1 ng/ml granulocyte–macrophage colonystimulating factor (GM-CSF) (Leucomax; Schering-Plough).
When indicated, cultures were incubated with 5 ␮g/ml anti–
BDCA-2 (clone AC144) or anti–BDCA-4 (clone AD5-17F6)
monoclonal antibodies (mAb) (Miltenyi Biotec, Bergisch
Gladbach, Germany). The mAb were dialyzed in order to
eliminate NaN3 content and were stabilized with 1 mg/ml HSA.
The PBMC cultures were then incubated at 37°C in 7% CO2 in
air for 20 hours. For flow cytometry, 2-ml volumes of PBMCs
(2 ⫻ 106/ml) were stimulated for 7 hours with HSV-1 in
flat-bottomed 24-well plates (Nunc). Brefeldin A (10 ␮g/ml;
Sigma-Aldrich, St. Louis, MO) was added to the cultures 2
hours before harvest in order to increase intracellular concentrations of IFN␣.
Immunoassays. The concentrations of IFN␣ in supernatants were determined by dissociation-enhanced lanthanide
fluoroimmunoassay (DELFIA) as described (37), with modifications. Briefly, microtiter plates (Maxi-Sorp; Nunc) were
coated with the anti-IFN␣ mAb LT27:293 (produced in our
laboratory), which detects the majority of IFN␣ subtypes but
not IFN␣2b, which is used to supplement the IFN␣ induction
cultures. After washing, the plates were blocked with a postcoating buffer. The sample or standard was coincubated in a
total volume of 0.1 ml for 1 hour with the europium-labeled
anti-IFN␣ mAb LT27:297 (produced in our laboratory) in the
LT27:293-coated plates. After washing, fluorescence was measured in a 1234 DELFIA Research Fluorometer (Wallac,
Turku, Finland), and data were analyzed with MultiCalc
software (Wallac). Samples and standard were calibrated
against the National Institutes of Health reference leukocyte
IFN␣ GA-23-902-530.
Flow cytometry. PBMCs were incubated with Fc receptor (FcR) blocking reagent (Miltenyi Biotec) and stained with
anti–BDCA-2–fluorescein isothiocyanate (FITC), anti–
BDCA-3–FITC, or anti–BDCA-4–phycoerythrin (PE) mAb
(all murine IgG1; Miltenyi Biotec) for 10 minutes at 4°C, as
recommended by the manufacturer. FITC- or PE-conjugated
murine IgG1 mAb were used as negative control (Dako,
Glostrup, Denmark). After two washes, the cells were resuspended in PBS and analyzed by flow cytometry. All washes of
cells were performed at 4°C in PBS containing 0.2 mg/ml HSA
and 1 mg/ml NaN3.
After staining for surface antigens as described above,
the PBMCs were stained for intracellular IFN␣ when indicated. The cells were first fixed in 1% (weight/volume) paraformaldehyde overnight, then washed once in PBS and permeabilized in PBS containing 0.2 mg/ml HSA and 0.1% (volume/
volume) Tween 20. After blocking with 2% normal mouse
serum for 20 minutes, cells were incubated for 60 minutes with
biotinylated anti-IFN␣ mAb LT27:295 (produced in our laboratory). After washing, cells were stained with streptavidin–PE
(Jackson ImmunoResearch, West Grove, PA). At least 105
cells (BDCA single stainings) or 5 ⫻ 105 cells (double stainings) were acquired and analyzed using a FACScan flow
cytometer and CellQuest software (BD Biosciences, San Jose,
CA). A forward scatter–side scatter gate was set to include live
PBMCs. The numbers of BDCA-expressing cells per ml of
blood were calculated by multiplying the percentage of BDCApositive cells in PBMCs (determined in the FACScan flow
cytometer) by the number of mononuclear cells per ml of
blood (determined using a Cell-Dyn 4000 flow cytometer
[Abbott, Abbott Park, IL]).
Statistical analysis. Differences between groups were
analyzed using the Mann-Whitney U test and the Wilcoxon
signed rank test. Correlation analyses were performed using
Spearman’s rank correlation. All analyses were performed
using Statview 5.0 software (SAS Institute, Cary, NC).
Reduced IFN␣ production by PBMCs from patients with SLE. PBMCs from SLE patients have a
reduced capacity to produce IFN␣ in response to viruses
(37). We investigated whether there was a similar deficiency in the IFN␣ production triggered by immune
complexes containing interferogenic DNA (SLE-IIF),
which is probably a relevant endogenous IFN␣ inducer
in SLE. Therefore, PBMCs from SLE patients and
healthy controls were stimulated in vitro by SLE-IIF
and, for comparison, by HSV-1. The IFN␣ production of
PBMCs from SLE patients was reduced compared with
that of PBMCs from healthy controls (median 24
units/ml versus 190 units/ml; P ⫽ 0.002) (Figure 1A). As
expected, HSV-1–induced IFN␣ production of PBMCs
from SLE patients was also lower than that of PBMCs
from healthy controls (median 380 units/ml versus 940
units/ml; P ⫽ 0.0007) (Figure 1B). Consequently, IFN␣
production induced by both the endogenous IFN␣ inducer and HSV-1 is deficient in PBMCs from SLE
Reduced numbers of BDCA-expressing cells in
SLE patients. Because of the low IFN␣ production by
SLE PBMCs, we determined the numbers of circulating
NIPCs/PDCs, measured as cells expressing the BDCA-2
and BDCA-4 markers. In addition, the number of
BDCA-3–expressing cells was determined. We found
that the frequencies of BDCA-2– and BDCA-4–
expressing cells in PBMCs of SLE patients (median
0.32% and 0.31%, respectively) were lower than those in
PBMCs of healthy controls (median 0.51% and 0.44%,
respectively); the difference was statistically significant
for BDCA-2 (P ⫽ 0.01), but not for BDCA-4 (P ⫽ 0.05)
(Figure 2A). The frequency of BDCA-3–positive cells
was also lower in SLE patients than in healthy controls
(median 0.06% versus 0.09%; P ⫽ 0.004).
In addition, the actual numbers of BDCA-2–,
BDCA-3–, and BDCA-4–expressing cells per ml of
blood appeared reduced in SLE patients compared with
healthy controls (Figure 2B) in a manner similar to that
of the frequencies in PBMCs (Figure 2A). The differ-
Figure 1. Interferon-␣ (IFN␣) production by peripheral blood mononuclear cells from systemic lupus
erythematosus (SLE) patients (shaded boxes) and healthy controls (open boxes) stimulated by A, SLE
serum containing an endogenous IFN␣-inducing factor (SLE-IIF) or B, herpes simplex virus type 1
(HSV-1). IFN␣ levels in 20-hour culture supernatants were determined by an immunoassay. Boxes show
the 25th and 75th percentiles. Horizontal lines within the boxes show the median. Bars above and below
the boxes show the 10th and 90th percentiles. Open circles show outlying values. n ⫽ number of donors
examined. Significance levels for differences between groups were computed by the Mann-Whitney U test.
ence was significant for BDCA-2 (P ⫽ 0.01) and
BDCA-3 (P ⫽ 0.01), but not for BDCA-4 (P ⫽ 0.06).
IFN␣ production by BDCA-2–expressing cells. In
order to investigate the IFN␣-producing capacity of
BDCA-2–expressing cells, PBMCs from 5 SLE patients
and 5 healthy controls were stimulated by HSV-1. After
staining for BDCA-2 and intracellular IFN␣, cells were
analyzed by flow cytometry. We found that the frequency of IFN␣-producing cells among PBMCs was
lower in the SLE patients (median 0.09%, range 0.01–
Figure 2. Expression of blood dendritic cell antigens (BDCAs) 2, 3, and 4 on peripheral blood mononuclear cells (PBMCs) from systemic lupus
erythematosus patients (shaded boxes) and healthy controls (open boxes). PBMCs were stained with anti–BDCA-2, -3, and -4 monoclonal
antibodies and analyzed by flow cytometry. Results are shown as A, percentage of BDCA-positive cells among PBMCs and B, number of
BDCA-positive cells per ml of blood. Boxes show the 25th and 75th percentiles. Horizontal lines within the boxes show the median. Bars above
and below the boxes show the 10th and 90th percentiles. Open circles show outlying values. n ⫽ number of donors examined. Significance levels
for differences between groups were computed by the Mann-Whitney U test.
Figure 3. Flow cytometric analysis of intracellular IFN␣ and cell surface BDCA-2 in PBMCs stimulated
for 20 hours by HSV-1. Results are shown for PBMCs of 1 representative healthy control (A) and 1
representative SLE patient (B). Phycoerythrin (PE)–conjugated anti-IFN␣ was plotted against fluorescein
isothiocyanate (FITC)–conjugated anti–BDCA-2 monoclonal antibodies. The percentage of IFN␣producing cells (PE–IFN␣ positive) among BDCA-2–expressing cells (FITC–BDCA-2 positive) was
approximated by dividing the number of cells in gate R2 by the sum of the numbers of cells in gates R1
and R2 and multiplying by 100. See Figures 1 and 2 for other definitions.
0.14%) than in the healthy controls (median 0.23%,
range 0.11–0.49%) (P ⫽ 0.03) (Figure 3). The mean
fluorescence intensity (MFI) of the BDCA-2 signal did
not differ between PBMCs from SLE patients and
PBMCs from healthy controls before culture, but it was
markedly reduced in all individuals after culture. This
reduction in BDCA-2 MFI was more pronounced for the
IFN␣-positive cells in PBMCs from SLE patients (median MFI 11, range 9–19) compared with healthy controls (median MFI 21, range 18–24) (P ⫽ 0.02). Only a
minority of the BDCA-2–expressing cells produced
IFN␣ in healthy controls (median 18%, range 8–49%),
approximated by dividing the number of cells in gate R2
by the sum of the numbers of cells in gates R1 and R2
and multiplying by 100 (Figure 3). The corresponding
proportion in SLE patients could not be estimated due
to the low BDCA-2 signal.
In order to further elucidate the relationship
between the initial number of BDCA-2– and BDCA-4–
expressing cells and the total IFN␣ produced in HSV1–stimulated cultures of PBMCs, the proportions of cells
expressing these antigens were plotted against the IFN␣
levels in the medium. Interestingly, the proportions of
both BDCA-2– and BDCA-4–expressing cells among
PBMCs correlated with IFN␣ production for SLE patients (Figures 4A and B), but not for healthy controls
(Figures 4C and D).
Effects of ligation of BDCA-2 and BDCA-4 on
IFN␣ production by PBMCs. Ligation of BDCA-2 molecules by mAb inhibits IFN␣ production by PDCs from
healthy blood donors, and in vivo administration of such
mAb has therefore been suggested as a new therapy in
SLE (34). However, the effects of the anti–BDCA-2 and
anti–BDCA-4 mAb on PBMCs from SLE patients have
never been investigated. We therefore coincubated anti–
BDCA-2 or anti–BDCA-4 mAb with SLE-IIF– or HSV1–stimulated PBMCs from SLE patients and healthy
controls. As can be seen in Figure 5, IFN␣ production
was always strongly inhibited when PBMCs were coincubated with anti–BDCA-2 mAb, and especially SLEIIF–induced IFN␣ production by PBMCs from both
SLE patients and healthy controls was almost completely inhibited (median 98% and 97%, respectively).
In contrast, the anti–BDCA-4 mAb only partially inhibited SLE-IIF–induced IFN␣ production by PBMCs from
SLE patients and healthy controls (median 70% and
57%, respectively) and failed to inhibit IFN␣ production
in HSV-1–stimulated cultures (Figure 5).
The ongoing IFN␣ production in SLE patients
can be an important etiopathogenic factor in the autoimmune process, and the NIPCs/PDCs may have a
pivotal role as the main IFN␣ producers (for review, see
refs. 31, 32, and 38). For this reason, different regimens
directed against IFN␣ have been proposed as therapeutic options in this disease, such as the administration to
SLE patients of neutralizing antibodies to IFN␣ (39) or
antibodies to the BDCA-2 molecules that are specifically
Figure 4. Scattergrams showing the relationship between HSV-1–induced IFN␣ production and the
percentages of BDCA-2–expressing cells (A and C) or BDCA-4–expressing cells (B and D) in PBMCs
obtained from SLE patients (A and B) or healthy controls (C and D). The IFN␣ levels in 20-hour culture
supernatants were determined by an immunoassay. The percentages of BDCA-2– and BDCA-4–positive
PBMCs were determined by flow cytometry before culture. n ⫽ number of individuals (see Figures 1 and
2 for other definitions).
expressed on NIPCs/PDCs (34). Anti–BDCA-2 antibodies abolished IFN␣ production by NIPCs/PDCs from
healthy individuals (34), but to our knowledge, there is
no relevant information regarding the BDCA-2 molecules in SLE. In the present study, we therefore examined the expression of BDCA molecules by NIPCs/PDCs
from SLE patients, and we investigated whether antiBDCA mAb could inhibit IFN␣ production in such cells
obtained from SLE patients to the same extent as in
NIPCs/PDCs from healthy controls.
We found that PBMCs from SLE patients produced IFN␣ when stimulated by SLE-IIF, one likely
endogenous IFN␣ inducer in SLE, but IFN␣ levels were
markedly reduced compared with those in healthy individuals. Such reduced IFN␣ production was also seen
with a viral inducer, HSV-1, confirming previous results
(37). The low but significant IFN␣ production induced
by SLE-IIF strengthens the hypothesis that such interferogenic immune complexes are relevant endogenous
inducers in SLE. This reduced IFN␣ production by
PBMCs from SLE patients could be due to a low
number of circulating NIPCs/PDCs, deficient IFN␣ production by these cells, or both.
We previously reported a reduced frequency of
circulating functionally active NIPCs, responding to
HSV-1, in SLE patients (37). Investigators in later
studies, however, have reported conflicting data concerning the numbers of circulating NIPCs/PDCs in SLE.
In adult patients with SLE, the CD11c⫺ DC subset was
almost normal in one study (40), which instead indicated
that the CD11c⫹ DCs were reduced by 80%. In contrast, the CD123⫹,HLA–DR⫹,CD11c⫺ NIPC/PDC
Figure 5. The effect of anti–BDCA-2 or anti–BDCA-4 monoclonal
antibodies (mAb) on IFN␣ production induced by SLE-IIF or HSV-1
in PBMCs from 8 SLE patients (shaded boxes) and 7 healthy controls
(open boxes). IFN␣ levels in 20-hour culture supernatants were
determined by an immunoassay and expressed as percentages of the
levels of IFN␣ produced in PBMC cultures without mAb. Boxes show
the 25th and 75th percentiles. Horizontal lines within the boxes show
the median. Bars above and below the boxes show the 10th and 90th
percentiles. Open circles show outlying values. Significance levels for
differences between groups were computed by the Mann-Whitney U
test. See Figures 1 and 2 for other definitions.
population in pediatric SLE patients was reported to be
reduced (41). To resolve this issue, we used antibodies to
the two novel markers BDCA-2 and BDCA-4 that detect
the NIPCs/PDCs (33). We found that the proportion of
BDCA-2/4–expressing cells in PBMCs from SLE patients was decreased by ⬃30%, and their absolute numbers in blood were decreased by ⬃56%; the latter figure
is therefore concordant with that previously observed for
PDCs identified as CD123⫹,HLA–DR⫹,CD11c⫺ (41).
The BDCA-3–expressing cell population, which corresponds to the CD1c⫺,CD11c⫹,CD123⫺ DCs (33), was
also reduced in the PBMCs from SLE patients in our
study. These cells are included in the
lin⫺,CD4⫹,CD11c⫹ DCs, previously reported to be
reduced in SLE patients (40). Consequently, several DC
subpopulations are reduced in the blood of SLE patients.
The observed reduction of circulating NIPCs/
PDCs in SLE patients can partly be explained by a
redistribution of NIPCs/PDCs into tissues, as previously
proposed (28,29). However, the decreased number of
circulating NIPCs/PDCs is not of sufficient magnitude to
fully explain the 87% and 60% reductions of IFN␣
produced by SLE-IIF– or HSV-1–stimulated cells, respectively. Therefore, the IFN␣-producing capacity of
the remaining circulating NIPCs/PDCs in SLE may be
impaired. In order to explore this possibility, we used
double staining for BDCA-2 and intracellular IFN␣ to
determine whether all or only a subpopulation of NIPCs/
PDCs produced IFN␣. Our results revealed that only a
minority of the BDCA-2–positive cells in PBMCs from
healthy controls produced IFN␣, indicating that not all
NIPCs/PDCs respond to the IFN␣ inducer HSV-1.
However, it was difficult to accurately determine the
proportion of IFN␣ producers, because the BDCA-2
expression of PBMCs from healthy controls was strongly
reduced after culture, in accordance with previous observations that NIPCs/PDCs lose BDCA-2 upon maturation in vitro (33).
The BDCA-2 expression of PBMCs from SLE
patients was even more reduced, which made it impossible to estimate the proportion of IFN␣-producing cells
in the BDCA-2–positive population. This more pronounced decrease of BDCA-2 expression in SLE patients was interesting and may be due to the exposure of
the NIPCs/PDCs to cytokines, such as type I IFN,
GM-CSF, and IL-3, that are present in serum in these
patients (6,42,43). These cytokines are known to increase IFN␣ production induced in vitro (19,44) and to
promote maturation of NIPCs/PDCs (22,45,46). We
speculate that such influence of the SLE NIPCs/PDCs
by cytokines in vivo can explain the correlation between
the initial frequency of BDCA-2/4–positive cells and the
total in vitro IFN␣-producing ability of PBMCs observed in SLE patients, but not in healthy controls, in the
present study. However, this issue needs to be further
Although we did not directly determine the frequency of IFN␣-producing cells stimulated by SLE-IIF
in the BDCA-2–positive population, we have previously
observed that 5–10-fold fewer NIPCs/PDCs are activated by this inducer compared with HSV-1 (19). Therefore, although the actual IFN␣ producers and NIPCs/
PDCs share some phenotypic characteristics such as
BDCA-2/4, there may be a functional heterogeneity
among NIPCs/PDCs. Thus, subpopulations of NIPCs/
PDCs may have unique properties that make them
responsive to certain IFN␣ inducers. For instance, the
induction of IFN␣ by SLE-IIF, but not by HSV-1, is
dependent on Fc␥R type II expressed on NIPCs/PDCs
The different drugs used in the therapy of SLE
might affect NIPC/PDC function and numbers in blood.
In a previous study, however, we observed no difference
between the virus-induced in vitro IFN␣ production by
PBMCs from treated or untreated SLE patients (37).
Furthermore, in the present study, the frequency of
BDCA-2/4–positive NIPCs/PDCs did not differ between
SLE patients treated and those not treated with corticosteroids, or between patients with and patients without
chloroquine or azathioprine treatment (results not
shown). In contrast, reduced IFN␣ production by
PBMCs was reported during corticosteroid provocation
in 4 healthy volunteers (48). It is therefore important to
further evaluate the effects of corticosteroids and other
therapeutic agents on NIPCs/PDCs in SLE patients,
because these cells obviously constitute a potentially
relevant therapeutic target.
In the present study, we demonstrated that anti–
BDCA-2 mAb efficiently blocked IFN␣ production triggered by both HSV-1 and SLE-IIF in NIPCs/PDCs from
SLE patients, indicating that NIPCs/PDCs from these
patients do express sufficient numbers of target BDCA-2
molecules. In addition, we confirmed previous findings
(34) that this ligation of BDCA-2 inhibits the IFN␣
production triggered by the same IFN␣ inducers in
PBMCs from healthy blood donors. Our data therefore
support the contention (34) that anti–BDCA-2 mAb can
be of therapeutic value in SLE, specifically targeting
NIPCs/PDCs and inhibiting ongoing IFN␣ production.
Compared with the anti–BDCA-2 mAb, the anti–
BDCA-4 mAb had a weaker inhibitory action and
caused only a partial inhibition of the IFN␣ production
induced by SLE-IIF. Whether the differing sensitivity of
the HSV-1– and SLE-IIF–induced IFN␣ production to
the anti–BDCA-4 mAb is due to differences in the signal
mechanisms used by these two inducers remains to be
determined. If so, anti–BDCA-4 mAb could also be
suitable as a therapeutic agent in SLE, more specifically
inhibiting the endogenous IFN␣ inducer responsible for
the ongoing IFN␣ production in SLE.
We conclude that SLE patients have major alterations in the circulating NIPC/PDC population identified by the novel BDCA-2/4 markers. The activation by
the endogenous IFN␣ inducer SLE-IIF could still be
dramatically inhibited by anti–BDCA-2 mAb and, to a
lesser extent, by anti–BDCA-4 mAb, supporting the view
that humanized forms of these mAb should be evaluated
as therapeutic agents in SLE.
We thank Lotta Sjöberg, Inger Ohlsson, Anne Riesenfeld, Lisbeth Fuxler, and Anders Perers for excellent technical
assistance. We also thank patients and members of the staff for
their participation in this study.
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expressions, production, markers, dendriticum, lupus, systemic, erythematosus, bdca, plasmacytoma, cells, interferon
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