Expression of the markers BDCA-2 and BDCA-4 and production of interferon-╨Ю┬▒ by plasmacytoid dendritic cells in systemic lupus erythematosus.код для вставкиСкачать
ARTHRITIS & RHEUMATISM 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 Foundation. 1 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: firstname.lastname@example.org. Submitted for publication November 11, 2002; accepted in revised form May 9, 2003. 2524 BDCA-2/4 EXPRESSION AND IFN␣ PRODUCTION IN SLE 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 2525 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 METHODS 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 2526 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 BLOMBERG ET AL 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). RESULTS 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 patients. 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- BDCA-2/4 EXPRESSION AND IFN␣ PRODUCTION IN SLE 2527 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. 2528 BLOMBERG ET AL 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). DISCUSSION 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 BDCA-2/4 EXPRESSION AND IFN␣ PRODUCTION IN SLE 2529 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 2530 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␣ BLOMBERG ET AL 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 investigated. 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 (47). 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 BDCA-2/4 EXPRESSION AND IFN␣ PRODUCTION IN SLE 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. 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