ARTHRITIS & RHEUMATISM Vol. 58, No. 6, June 2008, pp 1741–1750 DOI 10.1002/art.23515 © 2008, American College of Rheumatology Direct B Cell Stimulation by Dendritic Cells in a Mouse Model of Lupus Suigui Wan, Zhenhai Zhou, Biyan Duan, and Laurence Morel Objective. Dendritic cells (DCs) play a major role in regulating lymphocytes, including B cells, and defective DC functions have been implicated in lupus. The purpose of this study was to assess the contribution of DCs to B cell hyperactivity in the B6.Sle1.Sle2.Sle3 (B6.TC) murine lupus model. Methods. We compared the effects of B6 and B6.TC bone marrow–derived DCs on naive B cells cocultured in the presence of lipopolysaccharide (LPS), anti-CD40, or anti-IgM. We measured the proliferation, antibody production, and expression of activation markers and chemokine receptors for the B cells, as well as DC cytokine production. B cell proliferation was also assessed in Transwell experiments and in response to activated DC supernatants or exosomes. The role of DC-produced cytokines was evaluated with blocking antibodies and transgenic mice. Results. LPS-stimulated or anti-CD40–stimulated DCs from B6.TC mice increased B cell proliferation, antibody production, and chemokine receptor expression as compared with DCs from B6 mice. Cell-to-cell contact was not necessary for the augmented effect of the lupus-prone DCs. Anti-CD40 treatment induced a higher production of interleukin-6 (IL-6), soluble IL-6 receptor (sIL-6R), IL-10, and tumor necrosis factor ␣ in B6.TC DCs. Blocking these individual cytokines, however, did not abrogate the effects of B6.TC DCs. Addi- tional experiments also ruled out involvement of BAFF, IL-12, and interferon-␣. Conclusion. Activated DCs from B6.TC mice directly increase B cell effector functions. This effect depends on soluble factors released by activated DCs, but none of the single major DC-produced cytokines known to affect B cells are necessary. Increased sIL-6R production suggests that increased sensitivity to IL-6 may be involved. Lupus has been described as a disease of B cell hyperactivity (1), based on data accumulated both in patients and in mouse models of the disease, and this body of work has provided the rationale for the various B cell–targeting therapies currently under consideration (2). Intrinsic defects in B cell tolerance have been demonstrated in lupus-prone mice (3). Multiple studies, however, have shown that T cell help was necessary for B cell hyperactivity and the production of pathogenic autoantibodies (4). The role of other cellular components of the immune system in inducing the lupus B cell phenotypes has been given less attention. A growing number of studies concur in showing that dendritic cells (DCs) play a critical role in B cell development, mostly through the production of survival factors, such as BAFF, or cytokines, such as interleukin-12 (IL-12), IL-6, and interferon-␣ (IFN␣) (5). More recently, intravital microscopy has demonstrated direct cognate antigen transfer from DCs to B cells (6). DCs have been implicated in the pathogenesis of lupus by a number of studies, including one conducted in the B6.Sle1.Sle2.Sle3 (B6.TC) congenic mouse strain derived from the NZM2410 model (7). Increased numbers of DCs accumulate in the lymphoid organs of B6.TC and other lupus-prone mice (8–12). Abnormal costimulatory profiles have also been reported in DCs from lupus patients (13–15) and in (NZB ⫻ NZW)F1 (BWF1), NZM2410, and B6.TC mice (12,16). Injections of BWF1 syngeneic DCs primed with apoptotic cells Dr. Morel’s work was supported by NIH grants R01-AI045050 and R01-AI-058150. Suigui Wan, MD (current address: Xuanwu Hospital, Capital University of Medical Science, Beijing, China), Zhenhai Zhou, MD, Biyan Duan, PhD (current address: University of Texas Southwestern Medical Center, Dallas), Laurence Morel, PhD: University of Florida, Gainesville. Drs. Wan and Zhou contributed equally to this work. Address correspondence and reprint requests to Laurence Morel, PhD, Department of Pathology, Immunology and Laboratory Medicine, University of Florida, PO Box 100275, Gainesville, FL 32610-0275. E-mail: [email protected] Submitted for publication July 10, 2007; accepted in revised form February 25, 2008. 1741 1742 accelerated disease (17). Furthermore, injections of Sle3-expressing DCs into nonautoimmune C57BL/6 (B6) mice induced the production of anti-DNA antibodies (18). However, most of these studies have been interpreted as lupus-associated DCs producing altered signals and amplifying autoreactive specificities in T cells, which, in turn, provide help to autoreactive B cells. DCs can directly regulate B cells through the production of IFN␣, whose elevated production has been proposed to be at the core of lupus pathogenesis (19). IFN␣ lowers the B cell receptor threshold of activation (20) and accelerates differentiation into plasma cells (21). In contrast to humans with lupus, there is little evidence for a type I IFN signature in murine models of spontaneous disease (22). In fact, the Sle2 locus, which contains the Ifa gene cluster, corresponds to a lower IFN␣ production (23). We have, however, shown that DCs from B6.TC mice produce large amounts of IL-6 in response to lipopolysaccharide (LPS) (12) or anti-CD40 stimulation (the present study), and this cytokine prevents CD4⫹,CD25⫹ Treg cells from exerting their inhibitory function (12). This IL-6– dependent blockade of Treg function maps at least partly to Sle1a (24). IL-6 is a proinflammatory cytokine that induces B cell proliferation and differentiation into plasma cells (25,26). We therefore assessed the effect of B6.TC-derived DCs on B cell proliferation and differentiation. Our congenic experimental system allows the direct comparison of B6, B6.TC, and B6.Sle1a DCs on the same B cells, either B6-derived or B6.TC-derived. B6 and B6.TC mice share ⬎95% of their genome, including their major histocompatibility complex (MHC), B cell, and T cell receptor genes, and therefore represent a well-controlled experimental system. We show here that LPS- or anti-CD40–stimulated DCs from B6.TC mice supported a stronger B cell proliferation and antibody production than DCs from B6 mice. Furthermore, DCs from the lupus-prone mice induced an increased expression of the chemokine receptors that direct the B cells toward the follicles, T cell zones, and germinal centers. Cell-tocell contact between DCs and B cells increased overall B cell proliferation, but was not necessary for the differential effect of the lupus-prone DCs, and supernatant from activated B6.TC DCs was sufficient to induce a more robust B cell proliferation. Anti-CD40 treatment induced a higher production of IL-6, IL-10, and tumor necrosis factor ␣ (TNF␣) in DCs from B6.TC mice than in those from B6 mice. Blocking these individual cytokines, how- WAN ET AL ever, did not abrogate the increased effect of the B6.TC DCs. Additional experiments also ruled out involvement of BAFF, IL-12, and IFN␣. Activated B6.TC DCs also produced higher levels of soluble IL-6 receptor (sIL-6R), suggesting that IL-6 transsignaling may be involved in the lupus DC–B cell interactions. Our findings show that B6.TC DCs act directly on B cells to increase their effector function and that the Sle1a locus plays a major role in this phenotype. This effect depends on soluble factors produced by activated DCs, but none of the major DC-produced cytokines that are known to affect B cell growth and differentiation are necessary. MATERIALS AND METHODS Mice. The production of the triple congenic B6.NZM2410-Sle1.Sle2.Sle3 (B6.TC), B6.NZM2410-Sle1a (B6.Sle1a), and B6.NZM2410-Sle1.Sle2.Sle3.STAT-4 –/– (B6.TC.STAT-4–/–) mouse strains has been described previously (7,27,28). C57BL/6J (B6) mice were bred at the University of Florida at Gainesville. B6.129S6-Il6tm1Kopf (B6.IL-6–/–) and 129S1/SvImJ (referred to as 129) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice of the 129/Sv.IFNAR–/–strain (29) were a kind gift from Dr. Westley Reeves (University of Florida, Gainesville, FL). All mice used in our experiments were 2–3-month-old females. All experiments were conducted according to protocols approved by the Institutional Animal Care and Use Committee of the University of Florida. DC and B cell isolation. Single-cell suspensions of bone marrow (BM) cells were cultured in complete RPMI 1640 containing 10% fetal calf serum (FCS), 10 ng/ml of granulocyte–macrophage colony-stimulating factor, and 10 ng/ml of IL-4 (R&D Systems, Minneapolis, MN). Every 2 days, 50% of the culture medium was replaced with fresh medium. Cells were harvested after 6 days of culture, ascertained by flow cytometry, and then CD11c⫹ cells were positively selected with magnetic microbeads according to the manufacturer’s instructions (Miltenyi Biotec, Auburn, CA). Purity of the CD11c⫹ fraction was ⬎90%. Splenic B cells were purified by CD43 negative selection (B cell isolation kit; Miltenyi Biotec), and the purity of the B220high fraction was typically ⬎95%. CXCL13 (B lymphocyte chemoattractant [BLC]) production by DCs was assessed by quantitative reverse transcription– polymerase chain reaction (RT-PCR), as described by Ishikawa et al (9). Flow cytometry. Single-cell suspensions were treated with Fc receptor block (2.4G2) and then stained with monoclonal antibodies to mouse B220 (RA3-6B2), CD11c (HL3), CD80 (16-10A1), CD86 (GL1), CXCR4 (2B11), CXCR5 (2G8), or I-Ab (AF6-120.1) (all from BD PharMingen, San Diego, CA), or BAFF receptor (BAFF-R; clone 204406) or CCR7 (4B12) (both from R&D Systems), or isotype controls. Intracellular cytokine production was assayed by adding GolgiPlug (BD PharMingen) for the last 5 B CELL STIMULATION OF DCs IN MURINE LUPUS hours of stimulation. Cells were then stained with the appropriate antibodies to gate on the population of interest, and with anti–IL-6 (MP5-20F3; BD PharMingen) or BAFF (clone 121808; R&D Systems) after cell permeabilization. At least 50,000 cells were acquired per sample using a FACSCalibur cytometer (BD Biosciences, Mountain View, CA), and dead cells were excluded by forward and side scatter characteristics. ModFit software (Verity Software House, Topsham, ME) was used to analyze the 5,6carboxyfluorescein succinimidyl ester (CFSE; Invitrogen, Carlsbad, CA) dilution. DC and B cell cocultures. BM-derived DCs (2 ⫻ 104) from B6 or B6.TC mice were cocultured with 105 B cells from B6, B6.TC, B6.IL-6–/–, B6.TC.STAT-4–/–, or 129 mice in 96 round-well Costar plates (Sigma-Aldrich, St. Louis, MO) for 5 days in complete RPMI 1640 in the presence of either anti-IgM Fab (R&D Systems) or anti-CD40 (3/23; BD PharMingen) at 10 g/ml each or in the presence of 1 g/ml of LPS (SigmaAldrich). In some experiments, 2 ⫻ 104 DCs were stimulated first with anti-CD40 or LPS for 24 hours, and then the supernatant was added to 105 B cells, which were then cultured for 5 days. For Transwell experiments, 3 ⫻ 104 B cells were placed in the bottom wells and 1.5 ⫻ 104 DCs were placed in the top wells in the presence of anti-CD40 in both wells. After 5 days of culture, B cell proliferation was measured according to the CFSE dilution or with the CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI); 1 Ci of 3H-thymidine was added to each well for the last 16 hours of culture. In some assays, anti–IL-6 (MP5-20F3; BD PharMingen), anti–IL-10 (A5-4; BD PharMingen), or anti-TNF␣ (MP6-XT22; R&D Systems) blocking antibodies or isotype controls were added at 5 g/ml. After 72 hours of culture, IL-6, IL-10, IL-12, TNF␣, and sIL-6R (CD126) were measured in supernatants by sandwich enzyme-linked immunosorbent assay (ELISA) (R&D Systems). Exosomes were isolated as previously described (30). Briefly, DC supernatants were harvested and centrifuged at 300g for 10 minutes, 1,200g for 20 minutes, and 10,000g for 30 minutes to remove cell debris. Exosomes were then pelleted at 100,000g for 1 hour at 4°C and resuspended in cold phosphate buffered saline. The presence of exosomes was confirmed by measuring the protein concentration in the pelleted samples. Exosomes collected from the supernatant of 2 ⫻ 104 DCs were added to 105 B cells, which were then cultured as described above. IgM and IgG production after 4 days of coculture was measured by sandwich ELISA, as previously described (7), in supernatants diluted 1:10. All experiments were performed at least twice, and representative results are shown. Statistical analysis. Statistical analysis was performed with the GraphPad Prism 4 package (GraphPad Software, San Diego, CA). Data were compared using t-tests after verification of normal distribution. Dunnett’s tests were used when multiple comparisons were performed in the same experiment. P values less than or equal to 0.05 were considered significant. 1743 RESULTS Proliferation of, and production of antibodies by, naive B cells in the presence of activated DCs from lupus-prone mice. CD43– naive B cells from B6 mice proliferated significantly more when cocultured with anti-CD40–stimulated BM-derived DCs from lupusprone B6.TC than those from B6 controls (Figure 1A). We have previously shown that Sle1a results in increased DC activation (24). We therefore tested the effects of B6.Sle1a DCs on B cells, and the results were the same as with DCs from the B6.TC mice (Figure 1B), indicating that this locus plays a significant role in this phenotype. DCs from B6 (P ⫽ 0.006) and B6.TC (P ⫽ 0.026) mice increased B cell proliferation induced by B cell receptor cross-linking with anti-IgM. However, no difference was consistently observed between these 2 types of DCs, although in a few assays (see Figure 6E below), proliferation was increased with B6.TC DCs in the presence of anti-IgM. This result suggested that the differential proliferation was mediated by DCs that had been activated through CD40 signaling. This was confirmed by the fact that DCs from B6 and B6.TC mice did not show increased B cell proliferation over that of medium alone in the absence of anti-CD40 (Figure 1E) and that DCs from neither strain proliferated in response to antiCD40 (Figure 1D). As expected, B cells proliferated to anti-CD40 treatment alone (Figure 1E), but the presence of B6.TC DCs significantly enhanced the process. Direct evidence of increased B cell proliferation was obtained from CFSE dilution studies of B220⫹ gated cells after coculture in the presence of anti-CD40 and DCs from B6, B6.TC, or B6.Sle1a mice (Figure 1C). Similar results were obtained with B cells derived from B6.TC mice (Figure 1D). In the absence of DCs, B cells from B6.TC mice proliferated more to CD40 stimulation than did B cells from B6 mice (compare Figures 1C and D), but the addition of DCs resulted in a similar response in B cells from both strains. This result ruled out the possibility that the differential response to B6.TC DCs was due to alloreactivity between the 2 strains. No significant proliferation was observed in DCs from either B6 or B6.TC mice in response to anti-CD40 (data not shown). The increased ability of B6.TC DCs to induce B cell proliferation was not specific to stimulation through CD40 since LPS stimulation produced the same effect (Figure 1F). These data indicate that activation induces in B6.TC DCs an enhanced ability to promote B cell proliferation. 1744 WAN ET AL Figure 1. Induction of stronger B cell proliferation by activated dendritic cells (DCs) from B6.TC and B6.Sle1a mice. A and B, Induction of thymidine incorporation. B6.TC (TC) or B6.Sle1a (Sle1a) bone marrow–derived DCs induced greater thymidine incorporation in B6 B cells in the presence of anti-CD40 (␣CD40), but not anti-IgM (␣IgM), than did B6 DCs. C and D, Proliferation of B cells from B6 (C) or B6.TC (D) mice, as measured by the 5,6-carboxyfluorescein succinimidyl ester (CFSE) dilution in the last 4 divisions of B220⫹ gated cells in the presence of B6 or B6.TC DCs and anti-CD40. Representative flow cytometry plots are shown at the right. Shaded areas show the cell division peaks predicted by the ModFit software. Horizontal bars show the gate for CFSElow. E, Proliferation of B cells from B6 mice. DCs did not promote B6 B cell proliferation in the absence of anti-CD40 stimulation. Anti-IgM– and anti-CD40–stimulated B cells in the absence of DCs were used as controls. F, Proliferation of B cells from B6 mice with and without lipopolysaccharide (LPS; 1 g/ml) stimulation. LPS stimulation of DCs from B6.TC mice induced B6 B cells to proliferate significantly more than did those from B6 mice. Values are the mean and SEM of 4 mice per group. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01; ⴱⴱⴱ ⫽ P ⬍ 0.001 for the comparisons indicated or versus B6 DCs, by t-test. B CELL STIMULATION OF DCs IN MURINE LUPUS Figure 2. Induction of stronger antibody production by dendritic cells (DCs) from B6.TC mice in the presence of anti-CD40. Production of A, total IgM and B, total IgG by B cells from B6 mice after 4 days in culture with B6 or B6.TC (TC) DCs in the presence of anti-IgM (␣IgM) or anti-CD40 (␣CD40). OD ⫽ optical density (at 405 nm). C, Plasma cell differentiation B cells from B6 mice cocultured with B6 or B6.TC DCs, as measured by CD138 expression. D, B6.TC DC induction of a lower surface IgM expression on CD138⫹ B6 B cells in the presence of anti-CD40. Values are the mean and SEM of 4 mice per group. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01, by t-test. Naive B cells from B6 mice produced significantly more IgM and IgG antibodies in the presence of antiCD40–activated DCs than with B cell receptor crosslinking and DCs (Figures 2A and B). Anti-CD40– stimulated DCs from B6.TC mice showed significantly higher production of both IgM and IgG than those from B6 mice. The induction of plasma cell differentiation, as measured by CD138 induction, was not appreciably different in DCs from either strain (Figure 2C), but surface IgM expression on CD138⫹ cells was significantly reduced in the presence of B6.TC DCs (Figure 2D), indicating faster progression to terminal plasma cell differentiation. Chemokine receptors play a critical role in directing B cells toward the various microenvironments of the lymphoid tissues. CXCR5 ligation induces B cells to move into the follicles (31), and relative levels of CXCR5 and CXCR4 regulate movements within the germinal centers (32). CCR7 expression is required for B cells to enter the T cell zone (31). We found that anti-CD40–activated DCs from B6.TC mice induced 1745 significantly higher levels of CXCR5, CXCR4, and CCR7 expression on B6 B cells than on B6 DCs (Figures 3A, B, and C, respectively). Interestingly, B cells from B6.TC mice expressed higher levels of CXCR5 and CXCR4 than did B cells from B6 mice in the presence of either B6 or B6.TC DCs. Although DCs from old BWF1 mice secrete CXCL13 (9,33), we were not able to detect CXCL13 expression by quantitative RT-PCR in BM-derived DCs from young B6 or young B6.TC mice (data not shown). In addition, we have previously shown that SDF-1 levels were equivalent between B6 and B6.TC mice (34). Therefore, these 2 chemokines cannot account for the activation of B cells by B6.TC DCs. In contrast to chemokine receptors, coculture with DCs did not increase B cell activation, as shown for class II MHC in the present study (Figure 3D). Activation marker expression was higher on B cells from B6.TC mice than on those from B6 mice (as previously shown ), but it was similar with either B6.TC or B6 mouse DCs. Similar results were obtained with CD80 and CD86 stimulation (data not shown). DC stimulation of B cells through both contactdependent and contact-independent mechanisms. To assess whether the increased B cell proliferation induced Figure 3. Induction of increased expression of chemokine receptors, but not activation markers, on B6 B cells by dendritic cells (DCs) from B6.TC mice. The expression of A, CXCR5, B, CXCR4, C, CCR7, and D, class II major histocompatibility complex I-Ab, as determined by flow cytometry on B220⫹ gated B cells from B6 or B6.TC (TC) mice after 5 days of coculture with either B6 or B6.TC DCs, is shown. Values are the mean and SEM of 4 mice per group. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01, by t-test. 1746 WAN ET AL a significantly higher proliferation than supernatants from B6 DCs (Figure 4B). The same results were obtained with LPS-stimulated B6.TC DCs (data not shown). This effect was not due to DC-released exosomes present in these supernatants (Figure 4C and data not shown for exosomes extracted from LPS-activated DCs). In fact, exosomes, which have been shown to carry surface markers from the DC membrane (30), did not induce B cell proliferation above background, implying that soluble factors produced by activated B6.TC DCs are responsible for the enhanced effector function on B cells. These soluble factors do not involve the BAFF pathway, since intracellular production of BAFF did not differ between DCs from B6 and B6.TC mice after CD40 stimulation (mean ⫾ SEM 38.28 ⫾ 2.32% CD11c⫹,BAFF⫹ cells for B6 DCs versus 41.15 ⫾ 2.89% CD11c⫹,BAFF⫹ cells for B6.TC DCs) (Figure 5A). Similarly, BAFF-R expression on the B cell surface did not differ after culture with either B6 or B6.TC DCs (Figure 5B). Figure 4. Contact-independent enhancement of B cell proliferation by dendritic cells (DCs) from B6.TC mice. A, Proliferation of B cells from B6 mice after 5 days in simultaneous coculture with either B6 or B6.TC DCs mixed together (left) or separated in Transwells (right). B, Induction of B cell proliferation by anti-CD40 in supernatants and by direct cell contact. Supernatants from anti-CD40–stimulated B6.TC DCs induced B cell proliferation to a similar level as DCs cocultured with B cells in the presence of anti-CD40. Absorbance was read at 450 nm. C, Induction of B cell proliferation by anti-CD40 in exosomes and supernatants. Exosomes collected from anti-CD40–stimulated DCs failed to induce B cell proliferation, as compared with supernatants collected from the same cells. Values are the mean and SEM of 4 mice per group. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01; ⴱⴱⴱ ⫽ P ⬍ 0.001, by t-test. by DCs from B6.TC mice was contact dependent, we performed simultaneous Transwell and standard coculture experiments (Figure 4A). As expected, IgM stimulation in the presence of DCs was minimally reduced when contact between B cells and DCs was prevented. However, proliferation in the presence of anti-CD40 stimulation greatly decreased when contact was prevented between the B cells and DCs. This indicated that despite the fact that the known effect of anti-CD40– activated DCs on B cells is through the production of cytokines, cell-to-cell contact is important. However, the reduction was proportionally similar with DCs from both strains (mean ⫾ SEM proliferation in Transwells as the percentage of proliferation in conventional wells 45.07 ⫾ 4.17% for B6 DCs versus 39.48 ⫾ 3.99% B6.TC DCs; P ⫽ 0.18). Moreover, supernatants from B6.TC DCs activated for 24 hours with anti-CD40 still induced Figure 5. Lack of involvement of BAFF in the activation of B6.TC mouse dendritic cells (DCs) on mouse B cells. A, Representative intracellular BAFF staining relative to CD11c expression in B6 B cell cocultures with DCs from B6 or B6.TC (TC) mice. APC ⫽ antigenpresenting cells; PE ⫽ phycoerythrin. B, Surface expression of BAFF receptor (BAFF-R) on B220⫹ cells after 5 days of coculture in the presence of anti-CD40 (␣CD40). The histogram (right) shows representative BAFF-R expression on B220⫹ gated B6 (solid) or B6.TC (open) DCs. Horizontal bar shows the gate for BAFF-R⫹. FITC ⫽ fluorescein isothiocyanate. Values are the mean and SEM of 4 mice per group. B CELL STIMULATION OF DCs IN MURINE LUPUS In contrast, B6.TC DCs produced significantly more IL-6 than did B6 DCs after anti-CD40 stimulation of cocultures (Figure 6A). We further examined the origin of IL-6 by intracellular staining and showed that the B cells in the cocultures produced minimal amounts of IL-6, which was similar in the presence of DCs from either B6 or B6.TC mice (data not shown). Furthermore, the same response to anti-CD40–stimulated B6.TC DCs was similar in B cells from IL-6–deficient mice and B6 controls (data not shown), indicating that IL-6 did not act in an autocrine manner in our system. When IL-6 was neutralized with blocking antibodies at a concentration that was sufficient to abrogate the effect of B6.TC DCs on Treg cells (12), B6.TC DCs still induced greater proliferation than did B6 DCs (Figure 6E). Soluble IL-6R can enhance IL-6 signaling through a process known as trans-signaling (35), and increased IL-6 production has been shown to increase the production of sIL-6R (36). Interestingly, B6.TC DCs produced significantly more sIL-6R than did B6 DCs after anti-CD40 stimulation (Figure 6D), which could lower the B cell threshold response to IL-6. DCs from B6.TC mice also produced significantly more IL-10 and TNF␣ than did DCs from B6 mice after CD40 stimulation of cocultures (Figures 6B and C). Blocking IL-10 or TNF␣ significantly decreased B cell proliferation, but it did not abrogate the difference between B6.TC and B6 DCs (Figure 6F). Under our experimental conditions, IL-12, another DC-produced cytokine that regulates B cell differentiation, was produced in lower but similar amounts by DCs from B6 or B6.TC mice (data not shown). We tested the involvement of IL-12 and IFN␣ indirectly by using B cells from STAT-4–deficient (Figure 6G) and IFN␣/␤/ receptor 1 (IFNAR-1)–deficient (Figure 6H) mice, respectively. B cells from B6.TC.STAT-4–/– mice proliferated more than did B cells from B6.TC mice in the presence of anti-CD40– stimulated DCs from either B6 or B6.TC mice (Figure 6G). It is not clear at this point why coculture with B6.Sle1a DCs did not affect the proliferation of B6.STAT-4–/– B cells. To test the involvement of type I IFN, we used IFNAR-1–deficient mice, which have the 129/Sv genetic background. With both B6.TC and B6.Sle1a DCs, 129.Sv.IFNAR-1–/– B cells proliferated significantly more than did 129.Sv B cells as compared with B6 DCs. A similar result, although not statistically significant, was obtained with 129/Sv DCs. This latter control, as well as the fact that these experiments were conducted in Transwells, rules out alloreactivity as a mechanism for the increased proliferation. Overall, 1747 these results rule out IL-12 and IFN␣ as being responsible for the differential effect of B6.TC and B6 DCs on B cells. DISCUSSION The B6.TC congenic mouse strain is a model of lupus that contains the 3 NZM2410-derived major susceptibility loci on a nonautoimmune B6 background (B6.Sle1.Sle2.Sle3) (7). B6.TC mice accumulate DCs in their lymphoid organs with age, and these DCs result in higher proliferation of CD4⫹ T cells and inhibit the function of normal regulatory T cells (12). We have recently shown that DCs expressing Sle1a, one of the lupus susceptibility loci expressed in B6.TC mice, presented the same CD4⫹ T cell–activating phenotype as B6.TC DCs (24). In the present study, we found that B6.TC DCs directly contribute to B cell hyperactivity by increasing their proliferation, antibody production, and chemokine receptor expression. This phenotype was also present in Sle1a-expressing DCs. This indicates that Sle1a, which we initially characterized as a locus that contributes to lupus susceptibility through the production of autoreactive T cells (37), also contributes to lupus through the abnormal activation of T and B cells by DCs. The up-regulation of B cell activity by DCs from B6.TC or B6.Sle1a mice occurred in the presence of CD40 stimulation, which is critical for the differentiation and development of effector functions in B cells (38) and DCs (39). CD40L is overexpressed in B6.TC and B6.Sle1a CD4⫹ T cells (37). This suggests that reciprocal interactions occur between CD4⫹ T cells, DCs, and B cells in the B6.TC model, all of which contribute to enhanced effector phenotypes in both T and B lymphocytes. Our results also showed that the CD40 activation pathway is not unique and that B6.TC DCs activated by other stimuli, such as LPS, also stimulated B cells to a higher degree. We have previously shown that Toll-like receptor ligands induced a higher level of activation and cytokine production in DCs from B6.TC mice than in those from B6 mice (12). This suggests a mechanism by which subthreshold levels of activation may induce B6.TC DCs to activate B cells in vivo. Sle1a-expressing B cells express a higher level of activation markers (24,27), but we have not yet determined whether and to what extent it is B cell intrinsic. We have shown here that B6.TC or B6.Sle1a BMderived DCs, i.e., differentiated in the absence of T cells, up-regulated B cell functions, suggesting that these DCs have an abnormal intrinsic function. The results presented herein show that DC activation contributes to 1748 WAN ET AL Figure 6. Production of cytokines by mouse dendritic cells (DCs). DCs from B6.TC (TC) mice produced significantly more A, interleukin-6 (IL-6), B, IL-10, and C, tumor necrosis factor ␣ (TNF␣) than did DCs from B6 mice in the presence of B6 B cells and anti-CD40 (␣CD40), and produced more D, soluble IL-6 receptor (sIL-6R) in the presence of anti-CD40. This differential cytokine production was not responsible for the increased B cell proliferation, as shown in Transwell experiments in the presence of E, anti–IL-6 (␣IL-6) or F, anti–IL-10 or anti-TNF␣ (␣TNF␣) blocking antibodies. Ab ⫽ antibody. The involvement of IL-12 and interferon-␣ (IFN␣) was tested by comparing the responses induced by B6 and B6.TC DCs in G, B6.TC and B6.TC.STAT-4–/– B cells or H, 129/Sv and 129/SV.IFNAR-1–/– B cells in Transwell experiments. Results in G are expressed as the percentage change in B6.TC.STAT-4–/– relative to B6.TC B cell responses to B6, B6.TC, or B6.Sle1a (Sle1a) DCs; those in H are expressed as the percentage change in 129/Sv.IFNAR-1–/– relative to 129/Sv B cell response to 129/Sv, B6, B6.TC, or B6.Sle1a DCs. WT ⫽ wild-type. Values are the mean and SEM of 4 mice per group. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01; ⴱⴱⴱ ⫽ P ⬍ 0.001, by t-test. B CELL STIMULATION OF DCs IN MURINE LUPUS these effector phenotypes in B6.TC and Sle1a B cells. Using Transwell experiments, we have shown that cellto-cell contact enhanced the effect of both B6 and B6.TC DCs on B cells, but the enhanced effect of B6.TC DCs was contact independent. DCs release membranebound exosomes, which have been shown to perform a number of effector functions in vivo and in vitro (40,41). Exosomes from either anti-CD40– or LPS-activated DCs were not able to induce B cell proliferation in our system, but the supernatants from the same cells induced proliferation to a level similar to that in the cocultures of B cells and DCs. These results demonstrate that soluble factors released by activated B6.TC DCs enhance B cell effector phenotypes. Analysis of the cytokine production showed that B6.TC DCs responded differently from B6 DCs to the same anti-CD40 stimulation by producing more IL-6, TNF␣, and IL-10. Decreased production of IL-12 was observed, however, which corresponds to the defective IL-12 response of NZM2410 macrophages to LPS (42), but differs from Sle3-only expressing DCs (18). Blocking of any of these individual cytokines did not alter these effects. These results indicate either that several of these 4 cytokines are involved, but they can substitute for each other, or that an as-yet-undetermined soluble factor is responsible. The higher expression of sIL-6R by antiCD40–stimulated B6.TC DCs could be due to an autocrine effect of high IL-6 production by these cells (36). IL-6 trans-signaling has recently been shown to inhibit the induction of forkhead box P3 in naive CD4⫹ T cells (43), and evidence for its role in chronic inflammation and autoimmunity is accumulating (35). We hypothesize that in our specific model, sIL-6R increases B cell responses to IL-6, and we are currently testing this hypothesis. Myeloid cells have previously been shown to play a major role in disease pathogenesis in the NZM2410 lupus model through the expression of Sle3 (18). Sle3expressing DCs are associated with defective CD4⫹ T cell activation–induced cell death and the production of anti–double-stranded DNA antibodies. As we have shown here for B6.TC DCs, Sle3-expressing DCs produce high levels of proinflammatory cytokines (but not IL-6). In spite of these cytokines, the precise mechanisms by which either Sle3 or B6.TC DCs induce B cells to produce autoantibodies (for Sle3) or to display enhanced effector functions (for B6.TC) have not been yet determined. For Sle3, a direct interaction between DCs and B cells has not been examined. For B6.TC, we have now shown that DCs directly regulate CD4⫹ T cell and B cell hyperactivity and that Sle1a plays a major role in the process. Consequently, DCs should be considered an 1749 integral part of lupus pathogenesis, at least in the NZM2410 model. Moreover, Sle1a is a locus that affects both the intrinsic phenotype of CD4⫹ T cells (31,37) and DCs (present study), and these DCs regulate the functions of both CD4⫹ T cells and B cells. The Sle1a congenic interval represents a 2.49–2.96 Mb NZM2410derived segment that contains a maximum of 19 genes (http://www.ensembl.org/Mus_musculus/). The same gene in this locus could be responsible for all these phenotypes in CD4⫹ T cells and DCs. 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