Phenotypic and functional deficiencies of monocyte-derived dendritic cells in systemic lupus erythematosus (SLE) patients
Marcus Köller,
Bettina Zwölfer,
Günter Steiner,
Josef S. Smolen and
Clemens Scheinecker
Department of Rheumatology, Internal Medicine III, Medical University of Vienna, General Hospital of Vienna, Währingergürtel 18-20, A-1090 Vienna, Austria
Correspondence to: C. Scheinecker; E-mail: clemens.scheinecker{at}meduniwien.ac.at
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Abstract
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Systemic lupus erythematosus (SLE) represents an autoimmune disease for which alterations of T cells, B cells as well as various antigen-presenting cell (APC) populations have been described. In order to better define APC-associated deficiencies, we analyzed morphologic, phenotypic and functional characteristics of in vitro-generated monocyte-derived dendritic cells (MoDC) from SLE patients as compared with healthy controls. Analysis of MoDC at different stages of maturation revealed substantial phenotypic and functional defects of MoDC from SLE patients as compared with healthy controls. In particular, we observed a significantly reduced up-regulation of MHC class II molecules on MoDC upon activation which correlated with disease activity scores and functional deficiencies in mixed lymphocyte reaction experiments. Our data imply a crucial role of APC in the immunological imbalance in SLE for foreign and self-antigen reactivity.
Keywords: SLE, dendritic cells, monocytes, toll-like receptors
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Introduction
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Systemic lupus erythematosus (SLE) is a systemic autoimmune disease with multi-organ involvement characterized by the development of autoreactive T and B cells with specificity for double-stranded DNA and other nuclear auto-antigens.
The systemic immune alterations in SLE suggest that the disease might, at least in part, be driven by alterations of antigen-presenting cells (APC), and in particular, dendritic cells (DC) which are crucial for the activation of naive T cells specific for protein antigens both in vitro (1) and in vivo (2). Moreover, DC are highly efficient in presenting both exogenous (1) and endogenous (3) antigens to T cells. In line with this, DC have been suggested to play a major role not only in the effective presentation of foreign antigens during infection but also in defining immunological self and maintaining peripheral tolerance in the steady state [reviewed in (4)].
Major alterations in DC homeostasis have previously been described in SLE patients, in particular, certain peripheral blood DC subsets were found to be substantially decreased in SLE patients as compared with healthy controls (58). The reason for low numbers of DC in the peripheral blood of SLE patients, however, remains unclear. Several explanations could be put forward, including (i) decreased output from the bone marrow, (ii) increased migration to tissue and/or secondary lymphoid organs, (iii) increased apoptosis rates and (iv) deficiencies during differentiation and activation.
In addition, various deficiencies in the APC capacity have been described for SLE patients (7, 912), which may contribute to both the increased susceptibility of SLE patients to various infectious diseases (13, 14) as well as to deficiencies in sustaining peripheral tolerance to self-antigens.
Here we tried to address the question whether defects in the differentiation and activation of monocytes into DC might account for reduced peripheral blood DC counts and dysfunction in antigen presentation. Using an in vitro model system we compared the differentiation of DC from peripheral blood CD14+ monocytes from SLE patients and healthy controls. Analysis of morphology, phenotype and functional capacity of monocyte-derived DC (MoDC) at different stages of differentiation revealed major phenotypic and functional deficiencies of MoDC in SLE patients that might help to explain mechanisms that are involved in the pathogenesis of the disease.
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Methods
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Antibodies
A variety of mAb/fluorochrome conjugates were used in the study: mAb to CD3 (SK7), CD19 (4G7), CD33 (P67.6), CD56 (MY31) and HLA-DR (L243) were obtained from Becton Dickinson (San Jose, CA, USA); mAb to CD14 (UCH-M1), CD80 (BB-1) and CD83 (HB15a) were obtained from Serotec (Oxford, UK); mAb to CD16 (3G8) and CD54 (84H10) were obtained from Coulter Corp. (Hialeah, FL, USA) and mAb to CD40 (5C3) and CD86 (IT2.4) were obtained from PharMingen (San Diego, CA, USA). Antibodies against Toll-like receptor (TLR)-2 (TL2.1) and TLR-4 (HTA125) were obtained from Ebioscience (San Diego, CA, USA). In all experiments, control mAb of the same IgG isotype were included.
Patients and healthy controls
The SLE patients' individual data are summarized in Table 1. SLE patients were randomly selected from our out-patient clinic. They all fulfilled the American College of Rheumatology criteria for SLE (15). Sex- and age-matched healthy volunteers served as a reference population. All patient samples were collected between 8:00 AM and 12:00 PM in the course of venipuncture for routine laboratory investigations. All specimens were obtained with the approval of the local ethics committee after informed consent had been obtained from the donors. In all patients, European Consensus Lupus Activity Measurement (ECLAM) disease activity scores were determined (16).
Preparation of samples
A total of 2030 ml of heparinized whole blood was collected and processed within 4 h of collection. Heparinized blood samples were used for isolation of PBMC by standard gradient centrifugation with Ficoll-Hypaque (Pharmacia, Uppsala, Sweden). PBMC were washed twice with PBS and used immediately for further analysis.
Cell separation
Monocytes.
CD14+ monocytes were separated by high-gradient magnetic sorting using the VARIOMACS technique (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). This method has been described in detail elsewhere (17). Briefly, PBMC were incubated with saturating concentrations of anti-CD14 super-paramagnetic micro-beads for 20 min on ice and washed twice in PBS containing 5 mM EDTA and 0.5% human serum albumin. Labeled cells were passed through a column in a strong magnetic field and positively enriched cells were eluted from the magnetic column.
T cells.
T cells were prepared from PBMC by incubation of the cells with saturating concentrations of purified mAb against CD14, CD33, CD16, CD56, CD19 and HLA-DR molecules for 15 min on ice. After washing, the cells were incubated with anti-mouse IgG-conjugated super-paramagnetic micro-beads for 15 min and passed over a magnetic column as described above. The negatively selected T cells were collected and >98% expressed CD3 by flow cytometric analysis.
Immunofluorescence staining procedure
Fifty microliters of freshly isolated or cultured cells (0.1 x 107 ml1 to 1 x 107 ml1) were incubated for 15 min at 4°C with FITC-conjugated, PE-conjugated or peridine chlorophyll protein-conjugated mAb.
Determination of cell morphology
Freshly isolated CD14+ monocytes and MoDC after 8 days of culture with granulocyte macrophage colony-stimulating factor (GM-CSF) + IL-4 were centrifuged onto microscope slides using a Cytospin-2 centrifuge (Shandon Southern Products, Astmoor, UK), stained with MayGrünwaldGiemsa solution and analyzed by invert light microscopy (Olympus, Tokyo, Japan).
Cultivation of CD14+ monocytes
Isolated CD14+ cells were cultured at a cell density of 5 x 105 cells ml1 in standard culture flasks (Costar, Cambridge, MA, USA) in RPMI 1640/10% FCS-containing medium at 37°C in a humidified CO2-containing atmosphere. For induction of cell differentiation, combinations of recombinant human (rh) cytokines were used. RhGM-CSF was used at 50 ng ml1 and rhIL-4 at 1000 U ml1 for 8 days followed by LPS at 10 µg ml1 for 2 days.
Proliferation assays
For induction of mixed lymphocyte reaction (MLR), graded numbers of irradiated (3000 rad, 137Cs source) freshly isolated monocytes, immature MoDC or mature MoDC were incubated with 5 x 104 allogeneic T cells. For presentation of specific antigens, APC were incubated with autologous T cells in the presence of 5LF tetanus toxoid (TT, Connaught Laboratories, Willowdale, Ontario, Canada) or 100 µg ml1 of keyhole limpet hemocyanin (KLH). Cultures were performed in RPMI 1640 (BioWhittaker) supplemented with 10% FCS. Proliferation of T cells was monitored by measuring [methyl-3H]thymidine (Amersham, Buckinghamshire, UK) incorporation on day 6 of culture. All values of counts per minute were calculated from duplicates.
Apoptosis
MoDC were analyzed for proportions of apoptotic cells after 8 days of culture with GM-CSF + IL-4 and after an additional 2 days of culture in the presence of LPS (day 10). Apoptosis was assessed by two flow cytometric methods: phosphatidylserine staining by annexin V conjugated to FITC and staining for fragmented DNA by propidium iodide expression.
Flow cytometry
Flow cytometric analyses were performed with a FACScan flow cytometer (Becton Dickinson) equipped with a single laser emitting at 488 nm.
Statistical analysis
Student's t-test was used to determine whether the difference between control and sample was significant (P
0.05 being significant). For the assessment of correlations between disease activity and phenotype of MoDC, the Pearson's correlation coefficient was determined.
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Results
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Induction of DC differentiation of monocytes from SLE patients and healthy controls
Analysis of morphology.
CD14+ monocytes from SLE patients and healthy controls were isolated, analyzed and cultured in the presence of GM-CSF and IL-4. Cultures were set up with GM-CSF and IL-4 for 8 days in order to induce the differentiation of immature DC and for an additional 2 days in the presence of LPS in order to generate mature DC (1821). Analyses were performed before and after 8 days of culture (day 8), and after the additional 2 days of culture in the presence of LPS (day 10). As can be seen from Fig. 1, freshly isolated monocytes from SLE patients and healthy controls showed a similar morphology. After 8 days of culture both MoDC from SLE patients and healthy controls were found to be increased in size and the majority of cells developed typical dendritic cytoplasmatic extensions. A similar morphology was observed for both MoDC from SLE patients and healthy controls at day 10 of culture in the presence of LPS (data not shown).

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Fig. 1. Monocyte and MoDC morphology. Cytochemical staining of cell preparations. Representative photographs of MayGrünwaldGiemsa-stained cytospin preparations of freshly isolated CD14+ monocytes from healthy controls (a) and SLE patients (b) and MoDC after 8 days of culture with GM-CSF + IL-4 from healthy controls (c) and SLE patients (d).
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Phenotypic analysis.
As can be seen in Fig. 2, freshly isolated monocytes from both SLE patients and healthy controls were found to be HLA-DR+, CD14+, CD33+, CD54+ and CD86+ as determined by FACS analysis (black bars). A small proportion of cells were CD40+ and virtually all cells were negative for CD80 and CD83 expression. Comparative analysis of mean fluorescence intensity (MFI) values revealed similar expression levels for both monocytes from SLE patients and healthy controls for HLA-DR and CD14 and low levels for CD33, CD54 and CD86 (Fig. 3, black bars). After 8 days of DC differentiation in the presence of GM-CSF + IL-4, the majority of MoDC from both SLE patients and healthy controls was CD14 negative. A decrease was observed in CD33+ and CD86+ cells whereas the proportions of CD40+ cells increased substantially. A minor increase was further observed in proportions of CD54+ and CD80+ cells. Changes in MFI values from days 0 to 8 of culture were observed for several marker molecules, in particular for CD14 (decrease), CD40, CD54 and CD80 (increase), but were found to be similar for MoDC from SLE patients and healthy controls (see Fig. 3, hatched bars). During the final maturation of DC from days 8 to 10, however, compared with baseline, MHC class II (HLA-DR) molecule expression increased significantly (P = 0.04) on MoDC from healthy controls but not from SLE patients (Fig. 3, dotted bars), while the percentage of HLA-DR+ cells did not differ (Fig. 2, dotted bars). MFI changes of other surface marker molecules did not differ significantly.

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Fig. 2. Phenotypic analysis of monocytes and MoDC, I. Freshly isolated monocytes (filled bars), MoDC at day 8 of culture with GM-CSF + IL-4 (hatched bars) and MoDC at day 10 of culture upon addition of LPS for 2 days (dotted bars) of healthy controls (a) and SLE patients (b) were analyzed for the expression of surface marker molecules by FACS. Bars represent mean values ± SEM of proportions of positive cells (n = 25).
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Fig. 3. Phenotypic analysis of monocytes and MoDC, II. Freshly isolated monocytes (filled bars), MoDC at day 8 of culture with GM-CSF + IL-4 (hatched bars) and MoDC at day 10 of culture upon addition of LPS for 2 days (dotted bars) of healthy controls (a) and SLE patients (b) were analyzed for the expression intensity of surface marker molecules by FACS. Bars represent MFI values ± SEM of the surface marker molecules (n = 25).
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Stimulatory capacity of monocytes from SLE patients and healthy controls
Freshly isolated monocytes from healthy controls and SLE patients were analyzed for their capacity (i) to stimulate the proliferation of autologous T cells (a) without the addition of foreign antigens [autologous mixed lymphocyte reaction (AMLR)], (b) in a primary immune response upon addition of the foreign antigen KLH and (c) in a secondary immune response upon addition of the foreign antigen TT and (ii) to induce the proliferation of allogeneic T cells (allo-MLR). As can be seen from Fig. 4, freshly isolated CD14+ monocytes from healthy controls and SLE patients were comparable in their capacity to induce T cell proliferation in an AMLR, in a TT-specific or KLH-specific MLR and in an allo-MLR (Fig. 4, day 0 series).

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Fig. 4. T cell stimulatory capacity of monocytes and MoDC in the AMLR, antigen-specific MLR and allogeneic MLR (ALLO). For induction of the AMLR (or allogeneic MLR), T cells (5 x 104) were incubated with graded numbers of autologous (AMLR) or allogeneic (ALLO) irradiated monocytes (first column, day 0), MoDC at day 8 of culture with GM-CSF + IL-4 (second column, day 8) and MoDC at day 10 of culture upon addition of LPS for 2 days (third column, day 10) of healthy controls (filled squares) and SLE patients (empty circles). For presentation of specific antigen, cultures were set up in the presence of KLH or tetanus toxoid (TET.TOX). Proliferation of T cells was monitored in all instances by measuring [methyl-3H]thymidine incorporation on day 6 of culture. Degree of proliferation is indicated as counts per minute on the ordinate. The experiments represent mean values ± SEM calculated from duplicates (n 20).
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Stimulatory capacity of immature (day 8) MoDC from SLE patients and healthy controls
Upon induction of DC maturation with GM-CSF + IL-4 for 8 days, immature MoDC from healthy controls and SLE patients were comparable in their capacity to induce proliferation of autologous and allogeneic T cells in the absence of added antigen. Immature MoDC from SLE patients, however, compared with immature DC from healthy controls, induced a significantly lower proliferation of T cells in the TT-specific (P < 0.02) and KLH-specific (P < 0.02) MLR (Fig. 4, day 8 series).
Stimulatory capacity of mature (day 10) MoDC from SLE patients and healthy controls
At day 10 of culture, after the addition of LPS to day 8 cultures in order to induce the final maturation of immature MoDC, mature MoDC from SLE patients induced a significantly lower proliferative T cell response in the AMLR (P < 0.02), the TT-specific MLR (P < 0.05) and the KLH-specific MLR (P < 0.02) when compared with immature MoDC from controls. Both mature MoDC from healthy controls and SLE patients, however, were comparable in their capacity to induce the proliferation of allogeneic T cells (Fig. 4, day 10 series).
Apoptosis during DC differentiation of monocytes from SLE patients and healthy controls
In order to exclude the selective loss of MoDC subsets during culture due to differences in apoptosis rates of monocytes from healthy controls and SLE patients (22, 23), MoDC cultures from healthy controls and SLE patients were analyzed for proportions of apoptotic cells. Apoptosis was determined by FACS staining for annexin V and propidium iodide expression on days 8 and 10 of culture. As can be seen from Table 2, both cultures of MoDC from healthy controls and SLE patients were found to contain similar proportions of annexin V+ and propidium iodide+ cells on days 8 and 10 of culture. Thus, there was no selective loss of MoDC from SLE patients as compared with healthy controls during DC differentiation in vitro.
Correlation of phenotypic and functional deficiencies of MoDC from SLE patients with disease activity
Disease activity of SLE patients was assessed by the ECLAM (16) score and was correlated with phenotypic characteristics of DC. As can be seen from Fig. 5, expression levels of MHC class II molecules (HLA-DR) on mature DC, but not on resting monocytes or immature DC (data not shown), were inversely correlated with disease activity scores (R = 0.67, P = 0.002) in SLE patients, i.e. the lower the expression of HLA-DR molecules, the higher the disease activity. MHC class II expression levels were only weakly inversely correlated with doses of glucocorticoids (R = 0.4, P = 0.1). No strong correlation, however, was observed for other surface marker molecules or the functional capacity of MoDC, as analyzed in MLR cultures, with disease activity scores (data not shown). We cannot, however, exclude the possibility that additional correlations might become evident within a larger patient cohort.

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Fig. 5. Correlation of MHC class II molecule expression with disease activity scores in SLE patients. The correlation between the MFI values for MHC class II molecules (HLA-DR) of MoDC at day 10 of culture with the disease activity was established by calculating the Pearson's correlation coefficient (R = 0.67, P = 0.002). The ECLAM score was used to assess disease activity in SLE patients (n = 17).
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Analysis of TLR-4 and TLR-2 expression during DC differentiation
In order to characterize mechanisms that might account for the diminished up-regulation of MHC class II molecules on mature MoDC from SLE patients in comparison to healthy controls, we analyzed the expression of TLR-4 and TLR-2 during DC differentiation. As can be seen from Fig. 6, both freshly isolated monocytes from healthy controls (Fig. 6a) and SLE patients (Fig. 6b) expressed TLR-4 and TLR-2. For both, healthy controls and SLE patients, the expression intensity of TLR-4 and TLR-2 decreased during differentiation to immature MoDC (day 8). The expression intensity of TLR-4 increased again upon stimulation with LPS on mature MoDC (day 10). The expression intensity of TLR-2 on immature (day 8) and mature (day 10) MoDC was comparable. On the other hand and in contrast to MoDC from healthy controls, MHC class II expression did not increase during DC differentiation on immature (day 8) and mature (day 10) MoDC from SLE patients.

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Fig. 6. Analysis of TLR-4 and TLR-2 expression during DC differentiation. Surface FACS staining for HLA-DR, TLR-4 and TLR-2 expression (solid line) on freshly isolated monocytes, immature MoDC (day 8) and mature MoDC (day 10) in comparison to isotype-matched negative control staining (dotted line). Representations of a healthy control (a) and an SLE patient (b) out of five experiments are shown.
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Discussion
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In the present study, we describe a defect in the differentiation/activation of MoDC in SLE. This conclusion is based upon the following observations which are mutually supportive: (i) in contrast to healthy controls, during activation, MoDC from SLE patients fail to up-regulate HLA-DR molecules on their cell surface compared with their parent monocytes and (ii) with increasing maturation, SLE MoDC have a reduced capacity to promote autologous MHC-associated T cell activation compared with control MoDC, while their capacity to activate allogeneic T cells remains similar.
Analysis of freshly isolated CD14+ monocytes did not reveal significant phenotypic differences between SLE patients and healthy controls. In particular, and in contrast to previous reports (24, 25), we observed similar expression levels of MHC class II (HLA-DR) molecules on monocytes from SLE patients and healthy controls. In support of this finding, but in contrast to previous studies on infant SLE patients (8), freshly isolated monocytes from adult SLE patients had a T cell stimulatory capacity similar to that of control monocytes. Also, there was no morphologic or major phenotypic difference. This, however, might be due to differences in patient cohorts (adult versus infant SLE patients), their respective disease activity levels and/or the purity of isolated cell populations.
Upon induction of DC differentiation with GM-CSF + IL-4, HLA-DR expression levels tended to be lower, although not statistically significant, on immature (day 8) MoDC from SLE patients as compared with healthy controls. More importantly, the remarkable increase of HLA-DR expression that was induced on immature MoDC from healthy controls upon LPS stimulation, and which is well known to occur during DC maturation (1821), was not seen on MoDC from SLE patients.
The degree of deficiency of HLA-DR expression on mature DC was correlated with disease activity. Although it could therefore be regarded a consequence of active disease, we wonder if it does not rather constitute an intrinsic factor involved in, and contributing to, higher disease activity, since monocytes isolated to serve as DC parent populations had virtually identical phenotypic, morphologic and functional characteristics, whether from SLE patients or healthy controls. Thus, it is tempting to speculate that defects in the regulations of MHC class II expression levels might represent an underlying mechanism that contributes to the control of disease activity in SLE.
The molecular mechanism for LPS-mediated APC activation is TLR-4-mediated signaling (26, 27). No selective deficiency, however, was observed for TLR-4 expression on immature SLE MoDC, suggesting that defects in TLR expression do not account for the inadequate response of these immature MoDC to LPS stimulation. Beyond this, however, we cannot exclude the possibility that quantitative differences in TLR-4 expression levels or downstream defects in TLR-4 signaling might contribute to the decreased HLA-DR expression on mature MoDC from SLE patients.
DC are thought to participate in the regulation of peripheral tolerance toward self-antigens (4) and have been shown to constitutively present tissue-restricted self-antigens in secondary lymphoid organs (28). The subsequent deletion of potentially autoreactive T cells and/or the suppressive influence of regulatory T cells, however, apparently prevent the initiation of an immune reaction against self-structures. In line with this, an enhanced presentation of self-antigens in tissue-draining lymph nodes has been observed in autoimmune diseases (28, 29). A deficient presentation of self-antigens in the steady state could diminish the required T cell unresponsiveness toward self-antigens. This is thought to be crucial in order to avoid a concomitant reaction against self-antigens during the antigenic re-challenge in the presence of strong adjuvants in the course of an infection.
The AMLR can be used to estimate the constitutive presentation of self-antigens under steady-state conditions in vitro (30, 31). The reduced stimulatory capacity of MoDC in the AMLR as observed here for SLE MoDC might simply be explained by reduced MHC class II expression levels. Alternatively, however, it might represent the equivalent of an inadequate presentation of self-antigens under steady-state conditions in vivo and thus could at least partially help to explain defects in peripheral self-tolerance mechanisms in SLE patients.
We also observed a defect of mature MoDC to elicit both a primary as well as a recall T cell response. Such a deficiency in antigen-presenting capacity may contribute to, and be responsible for, a defective reactivity to foreign pathogens. Patients with SLE, in fact, have a higher infection rate than the general population, in particular during phases of active disease, and it is estimated that at least 50% of them will suffer a severe infectious episode during the course of the disease (13). Mature DC are thought to be crucially involved in the MHC class II-restricted presentation of foreign antigens during the course of an infection (32). Any defect in the MHC class II-mediated antigen presentation substantially affects the effectiveness of the final immune response. The concentration of MHC class II proteins on the surface of APC has been shown to inversely correlate with the susceptibility to infection (33) and is one of the parameters affecting the intensity of the immune response (34). Thus, our observation of a significantly diminished T cell stimulatory capacity of MoDC from SLE patients as compared with MoDC from healthy controls in a primary and secondary antigen-specific MLR, together with a potentially disturbed effector T cell function (35), could help to explain the increased susceptibility of SLE patients to various infections.
The functional deficiency of mature MoDC from SLE patients, as described herein, essentially affects one of the capacities to present foreign and self-antigens. Such deficiency might as well be a general one, including alloantigen presentation. However, this was not observed here, suggesting that the SLE MoDC deficiency was not global. Nevertheless, in vitro studies are generally limited by their experimental setting and culture conditions. Alterations of peripheral blood cell homeostasis or functional cellular deficiencies in SLE patients have previously been attributed to certain serum components (8, 36), but likewise, intrinsic cellular defects have been described (37). Our data, obtained from experiments under defined in vitro conditions, suggest an intrinsic functional defect of MoDC, which was most pronounced during the differentiation from immature to mature DC. This, however, does not argue against additional effects of serum factors or environmental stimuli in vivo which might further contribute to the immunological defects observed in SLE patients. In addition, future experiments will have to define whether certain components of culture supernatants might interfere with the in vitro DC differentiation and/or activation of MoDC from SLE patients.
This study was initiated in order to further analyze alterations in peripheral blood DC of SLE patients as described previously (7). Conclusions, however, might be limited since MoDC, generated under defined in vitro conditions, cannot simply be regarded as the in vitro equivalent of myeloid peripheral blood DC. This might explain differences between MoDC and peripheral blood DC in SLE patients in phenotypic and functional analysis.
Today, SLE still represents an incurable disease. However, we strongly believe that the increasingly detailed knowledge about specific pathogenic defects and mechanism of the disease will ultimately help to develop therapies that aim to restore the imbalance in the immunological response toward self- and foreign antigens as one of the driving forces in SLE.
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Acknowledgements
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We thank Mrs Irene Radda for proofreading the manuscript. This work was supported by the Austrian Science Fund Grant No. P13628-MED.
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Abbreviations
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AMLR | autologous mixed lymphocyte reaction |
DC | dendritic cells |
ECLAM | European Consensus Lupus Activity Measurement |
GM-CSF | granulocyte macrophage colony-stimulating factor |
KLH | keyhole limpet hemocyanin |
MFI | mean fluorescence intensity |
MLR | mixed lymphocyte reaction |
MoDC | monocyte-derived dendritic cells |
rh | recombinant human |
SLE | systemic lupus erythematosus |
TLR | Toll-like receptor |
TT | tetanus toxoid |
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Notes
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Transmitting editor: M. Feldmann
Received 29 October 2003,
accepted 20 August 2004.
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