B cells play critical roles in the pathogenesis of lupus. To examine the influence of B cells on disease pathogenesis in a murine lupus model, New Zealand Black and New Zealand White F1 hybrid (NZB/W) mice were generated that were deficient for CD19 (CD19−/− NZB/W mice), a B cell-specific cell surface molecule that is essential for optimal B cell signal transduction. The emergence of anti-nuclear Abs was significantly delayed in CD19−/− NZB/W mice compared with wild type NZB/W mice. However, the pathologic manifestations of nephritis appeared significantly earlier, and survival was significantly reduced in CD19−/− NZB/W mice compared with wild type mice. These results demonstrate both disease-promoting and protective roles for B cells in lupus pathogenesis. Recent studies have identified a potent regulatory B cell subset (B10 cells) within the rare CD1dhiCD5+ B cell subset of the spleen that regulates acute inflammation and autoimmunity through the production of IL-10. In wild type NZB/W mice, the CD1dhiCD5+B220+ B cell subset that includes B10 cells was increased by 2.5-fold during the disease course, whereas CD19−/− NZB/W mice lacked this CD1dhiCD5+ regulatory B cell subset. However, the transfer of splenic CD1dhiCD5+ B cells from wild type NZB/W mice into CD19−/− NZB/W recipients significantly prolonged their survival. Furthermore, regulatory T cells were significantly decreased in CD19−/− NZB/W mice, but the transfer of wild type CD1dhiCD5+ B cells induced T regulatory cell expansion in CD19−/− NZB/W mice. These results demonstrate an important protective role for regulatory B10 cells in this systemic autoimmune disease.

Systemic lupus erythematosus (SLE) is a prototypic multisystem autoimmune disease characterized by the production of autoantibodies and the involvement of most organ systems (1). Recent studies have demonstrated a critical role for B cells in SLE pathogenesis (24). In addition to autoantibody production, abnormal B cell activities or functions, such as cytokine production and Ag presentation, are likely to contribute to SLE development. Indeed, B cell-targeted therapies including mAbs to CD20, CD22, and BAFF are currently under evaluation in the treatment of human SLE (58).

B cell activation depends on BCR-generated signals during immune responses to self and foreign Ags (9). Cell surface and intracellular molecules that inform B cells of their microenvironment, such as CD19, CD22, Fc receptors, and TLRs, also play critical roles in controlling B cell responses (10). Among these molecules, CD19 serves as a positive response regulator that amplifies the strength and duration of BCR and other signaling events by regulating Src-family protein tyrosine kinases, and other effector molecules (1119). CD19 is a 95-kDa member of the Ig superfamily and is expressed on B cells and potentially follicular dendritic cells. CD19−/− mice are hyposensitive to a variety of transmembrane signals (20, 21), whereas B cells from transgenic mice that overexpress CD19 are hyperresponsive to transmembrane signals and generate autoantibodies spontaneously (22, 23), suggesting that altered CD19 function or expression can influence B cell susceptibility to autoimmunity (24). Therefore, selective targeting of CD19 might be a less invasive B cell-directed strategy for treating SLE rather than total B cell depletion.

As a well-established murine lupus model, New Zealand Black (NZB) and New Zealand White (NZW) F1 hybrid mice (NZB/W mice) spontaneously develop as SLE-like disease in which IgG anti-dsDNA autoantibody production is associated with immune complex-mediated glomerulonephritis (25). Aged NZB/W mice have increased numbers of splenic CD23loCD21hi marginal zone B cells as well as increased numbers of peritoneal B220intCD5+ B1 cells, although their significance in the pathogenesis has been unclear (2629). Recent studies have identified a phenotypically unique subset of spleen regulatory B cells that share phenotypic markers with both B-1 and marginal zone B cells (3033). A portion of these rare CD1dhiCD5+ B cells are competent for IL-10 production and are therefore called B10 cells (34). B10 cells and potentially other regulatory B cell subsets negatively regulate inflammation and autoimmune disease in mice, including contact hypersensitivity, experimental autoimmune encephalomyelitis, inflammatory bowel diseases, and arthritis (3040). Both contact hypersensitivity responses and experimental autoimmune encephalomyelitis are augmented in CD19−/− mice because of the absence of B10 cells (34, 41, 42). Whereas B cells and autoantibodies play major pathogenic roles in NZB/W mice, B cells can also contribute to the suppression of the disease. In this context, we assessed the effect of CD19 deficiency on disease initiation and progression in NZB/W mice.

NZB, NZW, and C57BL/6 mice were purchased from Japan SLC (Shizuoka, Japan). CD19−/− mice were generated as described (21) and backcrossed onto a C57BL/6 genetic background ≥12 times. CD19−/− mice were also backcrossed 12 times onto the NZB or NZW genetic backgrounds to obtain CD19−/− NZB mice and CD19−/− NZW mice. Female NZB/W mice were generated by mating female NZB and male NZW mice. Female CD19−/− NZB/W mice were generated by mating female CD19−/− NZB and male CD19−/− NZW mice. Mice were housed in a specific pathogen-free barrier facility. All procedures were approved by the Animal Committee of International Medical Center of Japan.

Serum samples were obtained from NZB/W mice and CD19−/− NZB/W mice every 2 wk for determining serum IgG anti-nuclear Ab (ANA) levels. To determine ANA positivity, serum was diluted 1:100 and added to fixed HEp-2 cell ANA slides (MBL, Nagoya, Japan) with FITC-conjugated goat anti-mouse IgG (H+L; ICN Biomedical, Costa Mesa, CA) used as the indirect immunofluorescence detection reagent at predetermined optimal concentrations. Also, sera at 12, 20, 28, and 36 wk old were diluted 1:40, 80, 160, 320, 640, 1280, and 2560 to determine ANA titers, and were assessed as above. Immunofluorescence staining of the slides was evaluated on a fluorescent microscope at ×400 magnification. The serum levels of IgG anti-dsDNA Abs were measured using dsDNA-coated 96-well ELISA plates (Mesacup; MBL). Sera were diluted 1:100, added to the ELISA plates, and allowed to react for 1 h at room temperature. Subsequently, the plates were washed three times before adding predetermined optimal concentrations of HRP-conjugated anti-mouse IgG Ab (Cappel; MP Biomedical, Irvine, CA). Ab binding was evaluated using TMB substrate (Bethyl Laboratories, Montgomery, TX), with the reactions stopped using 1N H2SO4, and read at a wavelength of 450 nm. A high-titer serum was plated in serial dilutions on each plate for quantification. The OD units were determined arbitrarily by taking a ratio between the OD values obtained for the test sample and for the high-titer sample at the same dilution.

Proteinuria was evaluated using Nephrosticks L (Bayer Medical, Tokyo, Japan). Kidneys were harvested from NZB/W and CD19−/− NZB/W mice and then bisected. The specimens were either fixed in 4% formalin for routine histologic analysis with H&E and periodic acid Schiff (PAS) staining or flash-frozen in OTC compound (Sakura Fineteck, Torrance, CA) for the detection of glomerular immune-complex deposits. The H&E- and PAS-stained sections were scored for interstitial and glomerular disease, as described previously (43), in a blinded manner. Cryostat-cut tissue sections from frozen samples were fixed in acetone for 5 min, and were incubated with 10% normal rabbit serum in PBS (10 min, 37°C) to block nonspecific staining. The tissue sections were incubated sequentially (20 min, 37°C) with predetermined optimal concentration of FITC-conjugated goat anti-mouse IgG (H+L) Ab (ICN Biomedical). The stained sections were read on a fluorescent microscope at ×400 magnification, and images were captured with a constant exposure time of 0.5 s. Mean fluorescence was calculated from captured images. Three representative glomeruli per mouse were outlined, and mean pixel intensity was calculated with Adobe Photoshop (Adobe Systems, San Jose, CA).

B cells were purified from single cell splenocyte suspensions by removing T cells with anti–Thy-1.2 Ab-coated magnetic beads (Dynal, Lake Success, NY). B cell suspensions were always > 95% B220+, as determined by flow cytometric analysis. B cells were resuspended (2 × 107/ml) in RPMI 1640 medium containing 5% FCS at 37°C. The cells were stimulated with goat anti-mouse IgM Ab F(ab′)2 fragments (40 μg/ml; Cappel) and subsequently lysed in buffer containing 1% NP-40, 150 mM NaCl, 50 mM Tris-HCl (pH 8.0), 1 mM Na orthovanadate, 2 mM EDTA, 50 mM NaF, and protease inhibitors. Protein concentrations were determined by light absorbance at 280 nm. The obtained lysates were subjected to SDS-PAGE with subsequent electrophoretic transfer to nitrocellulose membranes. These membranes were incubated with anti-phospho Akt Ab (Ser473; Cell Signaling, Beverly, MA), anti-active ERK Ab (Promega, Madison, WI), or anti-active JNK Ab (Promega), followed by incubation with HRP-conjugated donkey anti-rabbit IgG Abs (Jackson ImmunoResearch Laboratories, West Grove, PA). These blots were developed using an ECL kit (Pierce, Rockford, IL). To verify the presence of equivalent amounts of protein in each lane, the blots were stripped and reprobed with anti-ERK2 Ab (Santa Cruz Biotechnology, Santa Cruz, CA).

After spleen B cells had been stimulated with goat anti-mouse IgM F(ab′)2 Ab and lysed as described above, the lysates were analyzed using ProFluor Src-Family Kinase Assays (Promega) according to manufacturer’s protocol. The lysates were mixed with Src-family kinase R110 substrate, with ATP added to initiate the kinase reaction. After incubating the plate at room temperature for 60 min, protease solution was added to each well and incubated for 60 min at room temperature. After terminating the protease reaction, the fluorescence of the liberated R110 was read at a wavelength of 525 nm. The fluorescence of each well inversely relates to kinase activity within the cell lysate. The kinase activity of wild type (WT) B cells stimulated for 3 min was defined as 100%.

Spleen cells (1 × 107/ml) in RPMI 1640 medium containing 5% BSA and 10 mM HEPES buffer were loaded with 1 μM Fluo-4 (Molecular Probes, Eugene, OR) at 37°C for 30 min. The cells were washed and stained with PE–Cy5-conjugated anti-B220 Ab for 20 min on ice and washed. The fluorescence ratio (525/405 nm) of B220+ cells was determined using an Epics Altra flow cytometer (Beckman Coulter, Miami, FL) with fluorescence intensity shown on a four-decade log scale. Fluorescence contours are shown as 50% log density plots. Positive and negative populations of cells were determined using nonreactive isotype-matched Abs (Southern Biotechnology Associates, Birmingham, AL) as controls for background staining. Baseline fluorescence ratios were collected in real time for 1 min before goat anti-mouse IgM F(ab′)2 Ab fragments (Cappel) were added. The results were plotted as fluorescence ratios at 10-s intervals, with increasing fluorescence ratios indicating increased intracellular calcium concentration.

Eight-week-old mice were immunized i.p. with 100 μg 2,4-dinitrophenylated keyhole limpet hemocyanin (DNP-KLH; LSL, Tokyo, Japan) in CFA and were boosted 21 d later with 100 μg DNP-KLH in IFA. The mice were bled before and after immunizations. Serum DNP-specific Ab titers were measured by adding diluted sera to ELISA plates coated with DNP-BSA (5 μg/ml) for 1 h at room temperature. After washing the plates five times, bound Abs was detected using HRP-conjugated goat anti-mouse IgM or anti-mouse IgG1 Ab (Southern Biotechnology Associates) at predetermined optimal concentrations. The ELISA plates were developed using TMB substrate (Bethyl Laboratories, Montgomery, TX), stopped with 1N H2SO4 and read at a 450-nm wavelength.

The following mAbs were used: FITC-, PE-, and PE-Cy5– conjugated anti-mouse B220 (CD45R, RA3-6B2; BD Pharmingen, San Diego, CA), FITC-conjugated CD19 (MB19-1; BD Pharmingen), FITC-conjugated CD1d (1B1; BD Pharmingen), PE-Cy5–conjugated CD4 (H129.19; BD Pharmingen), PE-conjugated CD5 (53-7.3; BD Pharmingen), and FITC-conjugated anti-Thy1.2 (30-H12; BD Pharmingen) mAbs.

Single-cell spleen suspensions were stained for two/three-color immunofluorescence analysis at 4°C using Abs at predetermined optimal concentrations for 20 min as described (14). Cell numbers were counted using a hemocytometer, with relative lymphocyte percentages among viable cells (based on scatter properties) determined by flow cytometric analysis. Erythrocytes were lysed after staining using FACS Lysing Solution (BD Biosciences, San Jose, CA). A PE-conjugated anti-mouse/rat/human FOXP3 Flow Kit (clone 150D; Biolegend, San Diego, CA) was used to detect intracellular Foxp3 expression by regulatory T (Treg) cells according to the manufacturer’s protocol. The labeled cells were analyzed on an Epics Altra flow cytometer (Beckman Coulter) with fluorescence intensity shown on a 4-decade log scale. Positive and negative populations of cells were identified using nonreactive isotype-matched Abs (Southern Biotechnology Associates) as controls for background staining.

Spleen B cells and T cells were purified with B220 mAb- and Thy1.2 mAb-coated microbeads (Miltenyi Biotech, Auburn, CA) by positive selection following the manufacturer’s instructions. In addition, CD1dhiCD5+ B cells were isolated from purified B cell preparations using an Epics Altra flow cytometer (Beckman Coulter) with purities of 85–95%. These cells were homogenized in Isogen S (Wako, Tokyo, Japan), with total RNA isolated according to the manufacturer’s instructions. Total RNA was reverse transcribed to cDNA using a Reverse Transcription System with random hexamers (Promega). Quantitative RT-PCR was performed using the TaqMan system (Applied Biosystems, Foster City, CA) with analysis using an ABI Prism 7000 Sequence Detector (Applied Biosystems) according to the manufacturer’s instructions. TaqMan probes and the primers for IL-10 and GAPDH were purchased from Applied Biosystems. Relative expression of the real-time PCR products was determined using the ΔΔCT technique. B cells from 28-wk-old WT NZB/W mice were used as the calibrator. Each set of samples was normalized using the difference in threshold cycle (CT) between the target gene and housekeeping gene (GAPDH): ΔCT = (CT target geneCT GAPDH). Relative mRNA levels were calculated by the expression 2–ΔΔCT, where ΔΔCT = ΔCT sample − ΔCT calibrator. Each reaction was performed in triplicate at the least.

Serum IL-10 levels were measured using mouse IL-10 ELISA kits (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. Diluted sera were added to a 96-well plate precoated with anti-mouse IL-10 Abs, incubated for 90 min at 37°C, and washed with buffer four times. After the addition of biotin-conjugated anti–IL-10 Ab, the plate was incubated for 45 min at 37°C. The plates were then washed and incubated with HRP-conjugated streptavidin for 45 min at 37°C. The ELISA was developed using stabilized chromogen, terminated with stop solution, and read at a wavelength of 450 nm.

IL-10 present in tissue culture supernatant fluid was also quantified using the same assays. Splenic B cells from WT NZB/W mice were purified with B220 mAb-coupled microbeads (Miltenyi Biotech). CD1dhiCD5+ B cells were isolated using an Epics Altra flow cytometer (Beckman Coulter). Isolated CD1dhiCD5+ B cells as well as CD5− B cells (3 × 105) were cultured in 200 μl RPMI 1640 medium containing 10% FBS, 10 mM HEPES, 55 μM 2-ME, 200 μg/ml penicillin, and 200 U/ml streptomycin (Life Technologies, Carlsbad, CA) in 96-well flat-bottom tissue culture plates at 37°C with 5% CO2 in the presence of LPS (10 μg/ml, Escherichia coli serotype O111: B4; Sigma-Aldrich, St. Louis, MO). After culture for 72 h, IL-10 concentrations in culture supernatant fluid were quantified. All assays were performed using triplicate samples.

Splenic B cells were purified using B220 mAb-coupled microbeads (Miltenyi Biotech) from 20-wk-old WT NZB/W mice. Spleen CD1dhiCD5+ B cells were isolated using an Epics Altra flow cytometer (Beckman Coulter) with purities of 85–95%. After isolation, 2 × 106 CD1dhiCD5+B220+ or CD5B220+ B cells were transferred i.v. into 20-wk-old CD19−/− NZB/W mice (n = 15), with nephritis and survival monitored. In some mice, spleen Treg cell numbers were assessed at 24 wk old.

ANA, proteinuria, and survival data were analyzed using Kaplan-Meier curves and the log-rank test. Unless indicated otherwise, comparisons between groups were made using the Mann-Whitney U test; p < 0.05 was considered statistically significant.

The age of ANA production in WT and CD19−/− NZB/W mice was compared using a fluorescent ANA assay with HEp-2 cells as substrates. Serum ANA was first detected in NZB/W mice between 16 and 24 wk old. However, the appearance of serum ANA was significantly delayed in CD19−/− NZB/W mice (p < 0.001; Fig. 1A). ANA titers were also significantly lower in CD19−/− NZB/W mice than in WT NZB/W mice at all the ages examined (Fig 1B). ANA staining had a homogenous to speckled nuclei staining pattern, with no difference observed between WT and CD19−/− mouse sera (data not shown). The development of autoantibodies to dsDNA was also delayed in CD19−/− NZB/W mice as determined by ELISA (Fig. 1C). Although most CD19−/− NZB/W mice eventually produced anti-dsDNA autoantibodies, their mean serum titers were significantly lower than those of WT NZB/W mice after 20 wk old. Consequently, CD19 expression positively regulates autoantibody production in NZB/W mice.

FIGURE 1.

CD19 deficiency attenuates ANA and anti-dsDNA Ab production in NZB/W mice. A, ANA positivity was determined in sera from WT and CD19−/− NZB/W mice that were collected every 2 wk. Sera were diluted 1:100 and ANA was detected by indirect immunofluorescence using HEp-2 cells. B, ANA titers and (C) anti-dsDNA Ab titers were determined in sera from WT and CD19−/− (19−/−) NZB/W mice at 12, 20, 28, and 36 wk old. ANA was determined in sequentially diluted sera by indirect immunofluorescence on HEp-2 cells. IgG anti-dsDNA Abs were measured in sera diluted 1:100 by ELISA. Serial dilutions of a high-titer serum are shown in the right panel. The OD units were determined arbitrarily by taking a ratio between the OD values obtained for the test sample and the high-titer sample at the same dilution. Each group contained 25 mice. *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 1.

CD19 deficiency attenuates ANA and anti-dsDNA Ab production in NZB/W mice. A, ANA positivity was determined in sera from WT and CD19−/− NZB/W mice that were collected every 2 wk. Sera were diluted 1:100 and ANA was detected by indirect immunofluorescence using HEp-2 cells. B, ANA titers and (C) anti-dsDNA Ab titers were determined in sera from WT and CD19−/− (19−/−) NZB/W mice at 12, 20, 28, and 36 wk old. ANA was determined in sequentially diluted sera by indirect immunofluorescence on HEp-2 cells. IgG anti-dsDNA Abs were measured in sera diluted 1:100 by ELISA. Serial dilutions of a high-titer serum are shown in the right panel. The OD units were determined arbitrarily by taking a ratio between the OD values obtained for the test sample and the high-titer sample at the same dilution. Each group contained 25 mice. *p < 0.05; **p < 0.01; ***p < 0.001.

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To assess renal disease in NZB/W mice, the relationship between proteinuria and IgG deposition in the basement membranes of glomeruli were first investigated. Protein levels >300 mg/dl in urine correlated with histologic nephritis based on H&E and PAS staining in both WT and CD19−/− NZB/W mice (Fig. 2A). Therefore, urinary protein excretion >300 mg/dl was defined as proteinuria onset. Proteinuria was monitored every 2 wk in WT and CD19−/− NZB/W mice. Proteinuria developed slightly but significantly earlier in CD19−/− NZB/W mice than WT NZB/W mice (p < 0.05 from 23 to 32 wk; Fig. 2C). Pathologic examination of the kidneys from 32-wk-old mice revealed that glomerulonephritis and interstitial nephritis developed both in WT and CD19−/− NZB/W mice. Glomerulonephritis and interstitial nephritis tended to be even more severe in CD19−/− NZB/W mice than WT mice (Fig. 2B, 2D). The deposition of IgG in the basement membrane of glomeruli was also observed in both WT and CD19−/− mice. The fluorescence intensity of glomerular IgG staining was also slightly higher in CD19−/− mice, although the difference was not statistically significant (Fig. 2B, 2D). Glomerular IgG deposition was even detected in the kidneys of CD19−/− NZB/W mice that had been found to be ANA negative (data not shown). Therefore, CD19−/− NZB/W mice developed glomerulonephritis earlier than did WT NZB/W mice, despite their low frequency and titers of anti-dsDNA Abs (Fig. 1).

FIGURE 2.

CD19 deficiency accelerates nephritis and shortens survival in NZB/W mice. A, The relationship between proteinuria levels and nephritis histopathology. Kidneys were harvested from NZB/W WT and CD19−/− mice with various levels of proteinuria and were fixed in 4% formalin for H&E and PAS staining. The sections were scored for interstitial (left panel) and glomerular (right panel) disease. Each group contained 20 mice. B, Kidneys from NZB/W WT and CD19−/− mice at 32 wk old were evaluated for interstitial and glomerular diseases (left panel) and glomerular IgG deposition (right panel). Mean glomerular fluorescence staining intensity (arbitrary units) was determined for quantification. Each group contained seven mice. C, Proteinuria (urinary protein excretion > 300 mg/dl) in WT and CD19−/− NZB/W mice was monitored every 2 wk. Each group contained 25 mice. D, Histopathologic analysis of nephritis. Representative kidney sections from WT and CD19−/− NZB/W mice were stained with H&E, PAS, or FITC-conjugated goat anti-mouse IgG (H+L) Ab for the detection of glomerular immune-complex deposits. Original magnification ×200. E, Survival of WT and CD19−/− NZB/W mice. Each group contained 25 mice. *p < 0.05.

FIGURE 2.

CD19 deficiency accelerates nephritis and shortens survival in NZB/W mice. A, The relationship between proteinuria levels and nephritis histopathology. Kidneys were harvested from NZB/W WT and CD19−/− mice with various levels of proteinuria and were fixed in 4% formalin for H&E and PAS staining. The sections were scored for interstitial (left panel) and glomerular (right panel) disease. Each group contained 20 mice. B, Kidneys from NZB/W WT and CD19−/− mice at 32 wk old were evaluated for interstitial and glomerular diseases (left panel) and glomerular IgG deposition (right panel). Mean glomerular fluorescence staining intensity (arbitrary units) was determined for quantification. Each group contained seven mice. C, Proteinuria (urinary protein excretion > 300 mg/dl) in WT and CD19−/− NZB/W mice was monitored every 2 wk. Each group contained 25 mice. D, Histopathologic analysis of nephritis. Representative kidney sections from WT and CD19−/− NZB/W mice were stained with H&E, PAS, or FITC-conjugated goat anti-mouse IgG (H+L) Ab for the detection of glomerular immune-complex deposits. Original magnification ×200. E, Survival of WT and CD19−/− NZB/W mice. Each group contained 25 mice. *p < 0.05.

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WT NZB/W mice begin to succumb to disease at ∼25 wk old (Fig. 2C), following the development of nephritis. In contrast, CD19−/− NZB/W mice begin to succumb to disease at ∼20 wk old, consistent with their accelerated proteinuria development. Median survival in CD19−/− NZB/W mice was significantly shorter in comparison with WT NZB/W mice (30 versus 35 wk; p < 0.05). Death in CD19−/− NZB/W mice followed the development of nephritis, although some mice did not have detectable ANA or minimal anti-dsDNA Abs (data not shown). Collectively, CD19 expression negatively regulates the development of renal disease, which accelerated mortality.

Because CD19 deficiency generally leads to an immunodeficient B cell phenotype in both mice and humans (20, 21, 44), the finding that CD19 deficiency accelerated disease progression in NZB/W mice was paradoxical. Therefore, it was determined whether strain differences might result in an unanticipated phenotype for B cells from CD19−/− NZB/W mice. Cell surface CD19 expression on B cells from the blood, spleen, and lymph nodes was identical between C57BL/6 and NZB/W mice (data not shown). In functional studies, IgM ligation generated augmented intracellular calcium responses by splenic B cells from NZB/W mice relative to C57BL/6 mice (Fig. 3A), which is consistent with previous reports of polyclonal B cell activation in NZB/W mice (4548). When WT and CD19−/− NZB/W B cells were compared, IgM-induced intracellular calcium responses were delayed in CD19−/− B cells (Fig. 3B), which is consistent with results obtained with CD19−/− B cells from C57BL/6 mice (49). IgM-induced Src-family kinase activation and Akt phosphorylation were also significantly reduced in CD19−/− NZB/W B cells compared with B cells from WT NZB/W mice (Fig. 3C, 3D), as previously reported for CD19−/− B cells from C57BL/6 × 129 mice (14, 50). Impaired ERK and JNK activation were also observed in CD19−/− NZB/W B cells compared with WT NZB/W B cells (Fig. 3D). The proliferation of B cells cultured in the presence of F(ab′)2 anti-IgM Abs was also reduced by CD19-deficiency in NZB/W mice (data not shown). In vivo, the influence of CD19 deficiency on humoral immune responses in NZB/W mice was assessed by immunizing mice with DNP-KLH, a T cell-dependent Ag. Following immunizations, the primary and secondary IgM and IgG1 responses in CD19−/− NZB/W mice were significantly lower than in WT NZB/W mice (Fig. 3E). Thus, CD19-deficiency in NZB/W mice results in B cell defects that are identical to those reported for CD19−/− mice on nonautoimmune backgrounds. This finding explains the impaired autoantibody production in CD19−/− NZB/W mice, but not the dissociated acceleration of nephritis progression.

FIGURE 3.

B cell responses to IgM ligation in CD19−/− NZB/W mice. A and B, Intracellular calcium concentration responses. Fluo-4–loaded splenic B cells from (A) WT C57BL/6 and WT NZB/W mice, and from (B) WT and CD19−/− NZB/W mice were stimulated with F(ab′)2 anti-IgM Abs at 1 min (arrow). The results were plotted as fluorescence ratios at 10-s intervals and represent three experiments. C, Src-family protein tyrosine kinase activity in splenic B cells from WT and CD19−/− NZB/W mice incubated with either medium alone (time 0) or with anti–IgM-Ab (40 μg/ml) for the indicated times before cell lysates were subjected to in vitro kinase assays. The kinase activity of wild type B cells stimulated for 3 min was defined as 100%. Values for each genotype group represent mean ± SEM results from three mice. *p < 0.05. D, Akt, ERK, and JNK phosphorylation after IgM ligation in B cells from WT and CD19−/− NZB/W mice. Splenic B cell lysates were obtained as in (C), subjected to SDS-PAGE, and transferred onto membranes. These membranes were incubated with Abs against pAkt, pERK, or pJNK. Anti-ERK blotting (bottom) serves as a loading control. These results are representative of those obtained in three independent experiments. E, T cell-dependent humoral immune responses in WT and CD19−/− NZB/W mice immunized with DNP-KLH on days 0 and 21 (arrows) and bled at the indicated times. Serum levels of anti-DNP Abs in five mice of each genotype were determined by isotype-specific ELISA. **p < 0.01.

FIGURE 3.

B cell responses to IgM ligation in CD19−/− NZB/W mice. A and B, Intracellular calcium concentration responses. Fluo-4–loaded splenic B cells from (A) WT C57BL/6 and WT NZB/W mice, and from (B) WT and CD19−/− NZB/W mice were stimulated with F(ab′)2 anti-IgM Abs at 1 min (arrow). The results were plotted as fluorescence ratios at 10-s intervals and represent three experiments. C, Src-family protein tyrosine kinase activity in splenic B cells from WT and CD19−/− NZB/W mice incubated with either medium alone (time 0) or with anti–IgM-Ab (40 μg/ml) for the indicated times before cell lysates were subjected to in vitro kinase assays. The kinase activity of wild type B cells stimulated for 3 min was defined as 100%. Values for each genotype group represent mean ± SEM results from three mice. *p < 0.05. D, Akt, ERK, and JNK phosphorylation after IgM ligation in B cells from WT and CD19−/− NZB/W mice. Splenic B cell lysates were obtained as in (C), subjected to SDS-PAGE, and transferred onto membranes. These membranes were incubated with Abs against pAkt, pERK, or pJNK. Anti-ERK blotting (bottom) serves as a loading control. These results are representative of those obtained in three independent experiments. E, T cell-dependent humoral immune responses in WT and CD19−/− NZB/W mice immunized with DNP-KLH on days 0 and 21 (arrows) and bled at the indicated times. Serum levels of anti-DNP Abs in five mice of each genotype were determined by isotype-specific ELISA. **p < 0.01.

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CD19 expression is critical for regulatory B10 cell development in C57BL/6 mice (34, 40, 41, 51). Therefore, the development of the spleen CD1dhiCD5+ B cell subset, which includes B10 cells, was assessed in NZB/W mice. A spleen CD1dhiCD5+B220+ B cell subset was identified in NZB/W mice that was increased in 28-wk-old WT NZB/W mice when compared with 12-wk-old mice (0.9 ± 0.2% at 12 wk and 2.3 ± 0.5% of B220+ cells at 28 wk). In contrast, splenic CD1dhiCD5+ B cells were virtually absent in CD19−/− NZB/W mice at both 12 and 28 wk old (0.07 ± 0.03% at 12 wk and 0.13 ± 0.03% at 28 wk; p < 0.05 versus WT mice at each equivalent age; Fig. 4A, Table I). CD19−/− NZB/W mice also had reduced numbers of splenic marginal zone B cells with a CD23loCD21hi phenotype as well as reduced numbers of peritoneal B1 cells with a B220intCD5+ phenotype (Table I), both of which increase with age in NZB/W mice (29).

FIGURE 4.

Decreased spleen CD1dhiCD5+ B cells and IL-10 production in CD19−/− NZB/W mice. A, CD1dhiCD5+ B cells in CD19−/− NZB/W mice. Splenocytes from 12- and 28-wk-old WT and CD19−/− NZB/W mice were stained with CD1d, CD5, and B220 Abs with flow cytometric analysis of the B220+ cells. The gates were set to delineate CD1dhiCD5+ B cells, with their relative frequency among total B cells indicated. The results are representative of those obtained from three independent experiments. B, IL-10 transcript expression by spleen B cells. RNA was isolated from CD1dhiCD5+B220+, CD5B220+ (other), and total B220+ B cells of WT and CD19−/− NZB/W mice at the indicated weeks of age. IL-10 mRNA levels were determined in quantitative RT-PCR assays and normalized relative to internal control GAPDH transcripts. Each value indicates mean (± SEM) results from three mice. *p < 0.05. C, IL-10 secretion by spleen B cells. CD1dhiCD5+B220+, CD5B220+ (other), and total B220+ B cells were isolated from WT and CD19−/− NZB/W mice at the indicated weeks of age as in B and cultured with LPS for 72 h. The concentration of IL-10 in tissue culture supernatant fluid was measured by ELISA. Each value indicates the mean (± SEM) results from three mice. *p < 0.05. D, Serum IL-10 levels from WT and CD19−/− NZB/W mice of the indicated ages were measured by ELISA. Each value indicates mean (± SEM) results from 12 mice. **p < 0.01.

FIGURE 4.

Decreased spleen CD1dhiCD5+ B cells and IL-10 production in CD19−/− NZB/W mice. A, CD1dhiCD5+ B cells in CD19−/− NZB/W mice. Splenocytes from 12- and 28-wk-old WT and CD19−/− NZB/W mice were stained with CD1d, CD5, and B220 Abs with flow cytometric analysis of the B220+ cells. The gates were set to delineate CD1dhiCD5+ B cells, with their relative frequency among total B cells indicated. The results are representative of those obtained from three independent experiments. B, IL-10 transcript expression by spleen B cells. RNA was isolated from CD1dhiCD5+B220+, CD5B220+ (other), and total B220+ B cells of WT and CD19−/− NZB/W mice at the indicated weeks of age. IL-10 mRNA levels were determined in quantitative RT-PCR assays and normalized relative to internal control GAPDH transcripts. Each value indicates mean (± SEM) results from three mice. *p < 0.05. C, IL-10 secretion by spleen B cells. CD1dhiCD5+B220+, CD5B220+ (other), and total B220+ B cells were isolated from WT and CD19−/− NZB/W mice at the indicated weeks of age as in B and cultured with LPS for 72 h. The concentration of IL-10 in tissue culture supernatant fluid was measured by ELISA. Each value indicates the mean (± SEM) results from three mice. *p < 0.05. D, Serum IL-10 levels from WT and CD19−/− NZB/W mice of the indicated ages were measured by ELISA. Each value indicates mean (± SEM) results from 12 mice. **p < 0.01.

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Table I.
Frequency and number of splenic B cell subsets in NZB/W mice
12 wk
28 wk
PhenotypeWTCD19−/−WTCD19−/−
Percentage of each B cell subset relative to B220+ cells 
CD21hiCD23lo 11.3 ± 2.5 1.5 ± 0.3* 17.0 ± 1.4 1.9 ± 0.4** 
B220loCD5+ 6.1 ± 1.1 2.1 ± 0.6* 7.1 ± 1.0 2.1 ± 0.8** 
CD1dhiCD5+ 0.9 ± 0.2 0.07 ± 0.03* 2.3 ± 0.5 0.13 ± 0.03* 
     
Number of each B220+ cell subset 
CD21hiCD23lo 5.7 ± 0.5 0.5 ± 0.1** 16.9 ± 3.3 8.8 ± 1.7* 
B220loCD5+ 3.1 ± 0.5 0.7 ± 0.2** 7.1 ± 1.7 0.9 ± 0.2* 
CD1dhiCD5+ 0.5 ± 0.1 0.02 ± 0.01* 2.2 ± 0.2 0.06 ± 0.01** 
12 wk
28 wk
PhenotypeWTCD19−/−WTCD19−/−
Percentage of each B cell subset relative to B220+ cells 
CD21hiCD23lo 11.3 ± 2.5 1.5 ± 0.3* 17.0 ± 1.4 1.9 ± 0.4** 
B220loCD5+ 6.1 ± 1.1 2.1 ± 0.6* 7.1 ± 1.0 2.1 ± 0.8** 
CD1dhiCD5+ 0.9 ± 0.2 0.07 ± 0.03* 2.3 ± 0.5 0.13 ± 0.03* 
     
Number of each B220+ cell subset 
CD21hiCD23lo 5.7 ± 0.5 0.5 ± 0.1** 16.9 ± 3.3 8.8 ± 1.7* 
B220loCD5+ 3.1 ± 0.5 0.7 ± 0.2** 7.1 ± 1.7 0.9 ± 0.2* 
CD1dhiCD5+ 0.5 ± 0.1 0.02 ± 0.01* 2.2 ± 0.2 0.06 ± 0.01** 

Values represent means (± SEM) results obtained from three mice of each genotype. Values represent the percentage of lymphocytes expressing the indicated cell surface molecules out of total B220+ lymphocytes, or B cell numbers calculated based on the total number of spleen lymphocytes.

*p < 0.05; **p < 0.01.

Because IL-10 production is the hallmark of B10 cells, IL-10 secretion by CD1dhiCD5+ B cells was investigated. At 12 wk old, IL-10 mRNA expression in splenic B cells was comparable between WT and CD19−/− NZB/W mice. IL-10 mRNA levels of splenic B cells from WT NZB/W mice were increased by 2.5-fold at 28 wk old compared with those at 12 wk (Fig. 4B, left). IL-10 mRNA levels in splenic B cells from CD19−/− NZB/W mice remained unaltered at 28 wk old. Whereas B10 cells are not only IL-10 secreting B cells in the spleen, increased numbers and enhanced activation of B10 cells can at least partially contribute to the increase of IL-10 expression in splenic B cells from WT mice, because CD1dhiCD5+ B cells from WT NZB/W mice produced augmented IL-10 levels at 28 wk compared with CD1dintCD5 B cells (p < 0.05; Fig. 4B, right). IL-10 secretion from B cells was 11.2-fold higher in WT mice by 12 wk old and 11.4-fold at 28 wk old, respectively, than in CD19−/− mice (p < 0.05 for each; Fig. 4C, left). When splenic B cells from WT NZB/W mice were separated into CD1dhiCD5+ cells and non-CD1dhiCD5+ cells, CD1dhiCD5+ cells secreted 4- to 5-fold more IL-10 compared with non-CD1dhiCD5+ B cells (p < 0.05; Fig. 4C, right). Thus, CD1dhiCD5+ B cells were increased in number and produced significant levels of IL-10 during disease, whereas these cells were severely reduced in CD19−/− NZB/W mice at all time points. In addition, serum IL-10 concentrations increased during disease progression in WT NZB/W mice, but remained significantly lower in CD19−/− NZB/W mice (p < 0.01 at 12, 20, and 28 wk; Fig. 4D). Therefore, modest IL-10 production and the absence of CD1dhiCD5+ B10 cells offered an explanation for accelerated disease in CD19−/− NZB/W mice.

To determine whether the absence of regulatory B10 cells in CD19−/− NZB/W mice explains their accelerated disease progression, CD1dhiCD5+B220+ B cells from 20-wk-old WT NZB/W mice were transferred into CD19−/− NZB/W mice of the same age. As a control, spleen CD5B220+ follicular B cells were also transferred into CD19−/− NZB/W mice. The transfer of WT CD1dhiCD5+B220+ B cells into CD19−/− NZB/W mice normalized nephritis onset (p < 0.05 at 23 wk; Fig. 5A) and prolonged survival until 35 wk old (p < 0.05; Fig. 5B) to the extent seen in WT NZB/W mice. In fact, CD19−/− NZB/W mice that received WT CD1dhiCD5+ B cells lived even longer than WT NZB/W mice (median survival, 37 versus 35 wk). Nephritis and survival were not significantly altered in CD19−/− NZB/W mice that received WT CD5B220+ cells. In addition, the adopted transfer of CD1dintCD5+ B cells did not improve nephritis or survival significantly (data not shown). Therefore, spleen regulatory CD1dhiCD5+ B cells can inhibit lupus progression when transferred into CD19−/− NZB/W mice, demonstrating that this subset normally inhibits disease initiation in NZB/W mice.

FIGURE 5.

WT CD1dhiCD5+ B cells delay nephritis onset and prolong survival in CD19−/− NZB/W mice. CD1dhiCD5+B220+ or CD5B220+ B cells (2 × 106) from 20-wk-old WT NZB/W mice were transferred into CD19−/− NZB/W mice of the same age, with subsequent monitoring of (A) proteinuria and (B) survival. All groups contained 15 mice. Nontreated WT and CD19−/− NZB/W mice assessed in Fig. 2 are shown as controls. *p < 0.05, for the period indicated by a horizontal bar.

FIGURE 5.

WT CD1dhiCD5+ B cells delay nephritis onset and prolong survival in CD19−/− NZB/W mice. CD1dhiCD5+B220+ or CD5B220+ B cells (2 × 106) from 20-wk-old WT NZB/W mice were transferred into CD19−/− NZB/W mice of the same age, with subsequent monitoring of (A) proteinuria and (B) survival. All groups contained 15 mice. Nontreated WT and CD19−/− NZB/W mice assessed in Fig. 2 are shown as controls. *p < 0.05, for the period indicated by a horizontal bar.

Close modal

CD4+Foxp3+ Treg cell numbers increase in WT NZB/W mice during disease (Fig. 6A) as described (52). The CD4+Foxp3+ Treg cell subset composed 2.4 ± 0.7% of splenic Thy1.2+ T cells (2.4 ± 0.8 × 106 cells) in 12-wk-old WT NZB/W mice. Treg cell frequencies increased to 8.5 ± 1.7% (17.0 ± 1.9 × 106 cells) in WT NZB/W mice that developed ANA and proteinuria at 28 wk old. Although spleen Treg cell frequencies were comparable between WT and CD19−/− NZB/W mice at 12 wk old (2.1 ± 0.8%; 1.8 ± 0.8 × 106 cells in CD19−/− mice), there was not a significant increase in the Treg subset in CD19−/− mice at 28 wk old (2.8 ± 0.9%; 2.8 ± 0.7 × 106 cells; p < 0.05 versus WT mice at 28 wk). IL-10 production is also a characteristic of Treg cells (53). Whereas T cells from 28-wk-old WT NZB/W mice expressed ∼10-fold higher IL-10 mRNA levels than did 12-wk-old mice, IL-10 production from splenic T cells was below detectable levels in CD19−/− NZB/W mice at 28 wk old (p < 0.05 versus WT mice; Fig. 6B). Thereby, Treg cell numbers and IL-10 production by splenic T cells was decreased in CD19−/− NZB/W mice. Because CD19 expression is restricted to the B cell lineage, these results suggest that B cells can influence Treg cell development and/or activation.

FIGURE 6.

Decreased Treg cells and IL-10 production in CD19−/− NZB/W mice. A, CD4+Foxp3+ regulatory T cells in CD19−/− NZB/W mice. Splenocytes from 12- and 28-wk-old WT and CD19−/− NZB/W mice were stained with anti-Thy1.2 and CD4 mAbs, followed by intracellular staining for Foxp3 and flow cytometric analysis. These results represent those obtained from three independent experiments. B, IL-10 transcript expression by spleen T cells from WT and CD19−/− NZB/W mice at the indicated weeks of age. IL-10 mRNA levels were analyzed by quantitative RT-PCR and normalized relative to the internal GAPDH control. Each value indicates mean (± SEM) results from three mice. *p < 0.05. C, CD1dhiCD5+ B cells from WT NZB/W mice increase CD4+Foxp3+ Treg cells in CD19−/− NZB/W mice. CD1dhiCD5+B220+ B cells (2 × 106) from 20-wk-old wild type NZB/W mice were transferred into CD19−/− NZB/W mice of the same age. Four weeks later, the numbers of spleen CD4+Foxp3+ Treg cells in CD19−/− NZB/W mice were assessed. WT and CD19−/− NZB/W mice of the same age were assessed as controls. Each value indicates mean (± SEM) results from seven mice. *p < 0.05.

FIGURE 6.

Decreased Treg cells and IL-10 production in CD19−/− NZB/W mice. A, CD4+Foxp3+ regulatory T cells in CD19−/− NZB/W mice. Splenocytes from 12- and 28-wk-old WT and CD19−/− NZB/W mice were stained with anti-Thy1.2 and CD4 mAbs, followed by intracellular staining for Foxp3 and flow cytometric analysis. These results represent those obtained from three independent experiments. B, IL-10 transcript expression by spleen T cells from WT and CD19−/− NZB/W mice at the indicated weeks of age. IL-10 mRNA levels were analyzed by quantitative RT-PCR and normalized relative to the internal GAPDH control. Each value indicates mean (± SEM) results from three mice. *p < 0.05. C, CD1dhiCD5+ B cells from WT NZB/W mice increase CD4+Foxp3+ Treg cells in CD19−/− NZB/W mice. CD1dhiCD5+B220+ B cells (2 × 106) from 20-wk-old wild type NZB/W mice were transferred into CD19−/− NZB/W mice of the same age. Four weeks later, the numbers of spleen CD4+Foxp3+ Treg cells in CD19−/− NZB/W mice were assessed. WT and CD19−/− NZB/W mice of the same age were assessed as controls. Each value indicates mean (± SEM) results from seven mice. *p < 0.05.

Close modal

To determine whether the absence of CD1dhiCD5+ B cells in CD19−/− NZB/W mice influences Treg cell expansion, CD1dhiCD5+ B cells from 20-wk-old WT NZB/W mice were transferred into CD19−/− NZB/W mice of the same age. Four weeks after cell transfers, the numbers and percentages of Treg cells in CD19−/− NZB/W mice given WT CD1dhiCD5+ B cells were significantly higher than in age-matched CD19−/− NZB/W mice (5.0 ± 1.1 × 106 cells versus 1.9 ± 0.3 × 106 cells; p < 0.05; 6.1 ± 0.1.2% versus 2.3 ± 0.2% of Th1.2+ T cells, p < 0.05; Fig. 6C). Therefore, B10 cells or other CD1dhiCD5+ regulatory B cells are likely to play a critical role in Treg cell expansion in NZB/W mice.

Nephritis and death were accelerated in CD19−/− NZB/W mice relative to WT NZB/W mice (Fig. 2), despite B cell hyporesponsiveness and their immunodeficient phenotype (Fig. 3) of CD19−/− mice (20, 21). These unexpected findings were due to the virtual absence of B10 cells in CD19−/− NZB/W mice (Fig. 4) as described previously for C57BL/6 CD19−/− mice (34, 51). This was confirmed by the adoptive transfer of splenic CD1dhiCD5+ B cells from WT NZB/W mice into CD19−/− NZB/W mice, which significantly prolonged their survival and demonstrated an important protective role for regulatory B10 cells in this systemic autoimmune disease. Consistent with these observations, B cell depletion by CD20 mAb treatment eliminated 99% of B10 cells and accelerated disease development in young NZB/W mice as demonstrated in the companion paper to these studies (54). These studies demonstrate protective roles for B cells in lupus pathogenesis.

CD19 expression had both protective and disease promoting roles in lupus pathogenesis in NZB/W mice. CD19 deficiency significantly delayed the generation of ANA, especially anti-dsDNA Abs, in this lupus-prone mouse strain (Fig. 1). However, the manifestation of nephritis was paradoxically accelerated by the loss of CD19, although the difference was modest (Fig. 2). This result paralleled enhanced mortality in CD19−/− NZB/W mice. This discrepancy mirrors the findings of Shi et al. (55) in transgenic mice that overexpress CD19 and express the Sle1 lupus susceptibility locus. In this case, CD19 overexpression augmented humoral autoimmunity, but did not accelerate mortality or clinical evidence of renal dysfunction. Consistent with this finding, B cells from these CD19-transgenic mice are hyper-responsive to transmembrane signals, but have significantly increased B10 cell numbers (21, 34, 51). Therefore, CD19 expression positively correlates with autoantibody production, but is likely to have opposing roles during autoimmune disease by regulating B10 cell development. That severe glomerulonephritis can occur in the absence of ANA, including anti-DNA Abs, and that autoreactive B cells can exert pathogenic effects independent of Ab secretion has also been demonstrated in other lupus-prone mouse strains (5658). Thus, the severe renal disease observed in CD19−/− NZB/W mice is likely to result from B cell functions other than autoantibody secretion. These studies demonstrate that this B cell function is attributable in part to the suppressive role of B10 cells that normally negatively regulate disease progression.

IL-10 is a pleiotropic cytokine with both immunosuppressive and immunostimulatory properties (53, 59). The role of IL-10 in lupus pathogenesis is complex, including the effects of high serum IL-10 levels in human SLE and lupus-prone mouse strains (6064). For example, serum IL-10 levels positively correlate with SLE disease activity scores and anti-dsDNA autoantibody titers, but negatively correlate with C3 and C4 levels and lymphocyte counts (60, 65, 66). Patients with SLE also have significantly more IL-10–secreting mononuclear cells in their peripheral blood than do normal controls, and disease severity correlates with increased numbers of circulating IL-10–secreting mononuclear cells (62). Furthermore, IL-10 production by B cells is higher for patients with SLE than in normal controls, and Ig production by SLE B cells is largely dependent on IL-10 (61). Therefore, IL-10 can be pathogenic for lupus acceleration, but may also be produced to reduce already existing autoimmune inflammation. Various treatments targeting IL-10 against SLE have also shown contradictory results. For example, IL-10 deficiency significantly enhances disease severity in MRL/lpr mice with increases in IFN-γ and IgG2a anti-dsDNA autoantibody production, which are suppressed by recombinant IL-10 treatment (67). In the current study, CD19 deficiency led to lower serum IL-10 levels in NZB/W mice throughout the disease course (Fig. 4D). In contrast, continuous anti–IL-10 mAb administration significantly delays disease development in NZB/W mice, which is attributed to increased TNF-α production (68). These contradictory findings are most likely explained by the fact that multiple cell types are capable of producing IL-10, including B cells. Thereby, the positive and negative regulatory roles of IL-10 are likely to differ depending on the cell source of IL-10, as well as the timing of its production, duration, and levels of IL-10 expression. Thus, B10 cell IL-10 production is but one component of a complex regulatory network that balances protective and pathogenic immune responses.

In addition to B10 cells and Ig secretion, B cells regulate immune responses through multiple mechanisms that have been described recently (69). B cells contribute to Ag-presentation, cytokine production, the regulation of lymphoid organogenesis, effector T cell differentiation, and dendritic cell function. It is also noteworthy that B cells have other critical roles in lupus, presumably through their interaction with T cells. For example, B cell deficiency in MRL/lpr mice results in the complete absence of inflammatory T cell renal infiltration (70). B cell ablation in MRL/lpr mice using CD79 mAb decreases the relative abundance of CD4 memory T cells and also reduces T cell infiltration into the kidneys (71). In contrast, MRL/lpr mice engineered to have B cells expressing surface-bound but not secretory Ig develop nephritis, which is characterized by renal T cell infiltration (56). Thus, B cells play pathogenic roles via cytokine secretion or Ag presentation (72). Because lupus develops under the complex regulation of different B cell subsets and their functions, the selective targeting of B cell subsets might lead to promising therapies for this and other autoimmune disorders.

Although the adoptive transfer of CD1dhiCD5+ B cells into CD19−/− NZB/W mice significantly improved survival, this treatment did not cure the underlying disease (Fig. 5). Because CD19-positive transferred cells were detected in the spleens of CD19−/− NZB/W mice 2 wk after injection, but not in 5 wk (data not shown), this may be partly explained by the eventual rejection of CD19-expressing WT B10 cells in CD19-deficient mice. However, this most likely reflects the complex etiology of the lupus-like diseases, and the involvement of multiple hematopoietic lineages in disease initiation and regulation. As an example, splenic T cell IL-10 mRNA levels were significantly reduced during the late stages of disease in CD19−/− NZB/W mice (Fig. 6B). The spleen CD4+Foxp3+ Treg cell subset was also significantly reduced in CD19−/− NZB/W mice, while Treg cells expanded during disease progression in WT NZB/W mice (Fig. 6A). Consistent with this finding, the adoptive transfer of CD1dhiCD5+ B cells from WT NZB/W mice significantly increased Treg cell numbers in CD19−/− NZB/W mice (Fig. 6C). These results indicate that CD19 expression by B cells or the presence or absence of B10 cells also has a significant influence on Treg cell development and/or activation in NZB/W mice that remains to be explored. Thus, effective treatments or a cure for lupus-like disease is likely to require the modulation of not only B cell and B10 cell functions, but also T cell and Treg cell functions that significantly modulate disease.

Disclosures T.F.T. is a paid consultant for MedImmune, Inc. and is a consultant and shareholder for Angelica Therapeutics, Inc.

This work was supported by a Grant-in-Aid from the Ministry of Education, Science, and Culture of Japan (to R.W. and M.F.) and Grants AI56363, CA105001, and CA96547 from the National Institutes of Health (to T.F.T.).

Abbreviations used in this paper:

ANA

anti-nuclear Ab

CT

threshold cycle

DNP-KLH

2, 4-dinitrophenyl-keyhole limpet hemocyanin

NZB

New Zealand Black

NZW

New Zealand White

NZB/W

New Zealand Black and New Zealand White F1 hybrid

PAS

periodic acids Schiff

SLE

systemic lupus erythematosus

Treg

regulatory T

WT

wild type.

1
Rahman
A.
,
Isenberg
D. A.
.
2008
.
Systemic lupus erythematosus.
N. Engl. J. Med.
358
:
929
939
.
2
Chan
O. T.
,
Madaio
M. P.
,
Shlomchik
M. J.
.
1999
.
The central and multiple roles of B cells in lupus pathogenesis.
Immunol. Rev.
169
:
107
121
.
3
Lipsky
P. E.
2001
.
Systemic lupus erythematosus: an autoimmune disease of B cell hyperactivity.
Nat. Immunol.
2
:
764
766
.
4
Anolik
J. H.
2007
.
B cell biology and dysfunction in SLE.
Bull. NYU Hosp. Jt. Dis.
65
:
182
186
.
5
Eisenberg
R.
,
Albert
D.
.
2006
.
B-cell targeted therapies in rheumatoid arthritis and systemic lupus erythematosus.
Nat. Clin. Pract. Rheumatol.
2
:
20
27
.
6
Dörner
T.
,
Lipsky
P. E.
.
2007
.
B-cell targeting: a novel approach to immune intervention today and tomorrow.
Expert Opin. Biol. Ther.
7
:
1287
1299
.
7
Ramos-Casals
M.
,
Soto
M. J.
,
Cuadrado
M. J.
,
Khamashta
M. A.
.
2009
.
Rituximab in systemic lupus erythematosus: A systematic review of off-label use in 188 cases.
Lupus
18
:
767
776
.
8
John Looney
R.
,
Anolik
J.
,
Sanz
I.
.
2009
.
A perspective on B-cell-targeting therapy for SLE.
Mod. Rheumatol.
20
:
1
10
.
9
Kurosaki
T.
2002
.
Regulation of B cell fates by BCR signaling components.
Curr. Opin. Immunol.
14
:
341
347
.
10
Tedder
T. F.
1998
.
Introduction: response-regulators of B lymphocyte signaling thresholds provide a context for antigen receptor signal transduction.
Semin. Immunol.
10
:
259
265
.
11
Carter
R. H.
,
Fearon
D. T.
.
1992
.
CD19: lowering the threshold for antigen receptor stimulation of B lymphocytes.
Science
256
:
105
107
.
12
Buhl
A. M.
,
Pleiman
C. M.
,
Rickert
R. C.
,
Cambier
J. C.
.
1997
.
Qualitative regulation of B cell antigen receptor signaling by CD19: selective requirement for PI3-kinase activation, inositol-1,4,5-trisphosphate production and Ca2+ mobilization.
J. Exp. Med.
186
:
1897
1910
.
13
Fujimoto
M.
,
Bradney
A. P.
,
Poe
J. C.
,
Steeber
D. A.
,
Tedder
T. F.
.
1999
.
Modulation of B lymphocyte antigen receptor signal transduction by a CD19/CD22 regulatory loop.
Immunity
11
:
191
200
.
14
Fujimoto
M.
,
Poe
J. C.
,
Jansen
P. J.
,
Sato
S.
,
Tedder
T. F.
.
1999
.
CD19 amplifies B lymphocyte signal transduction by regulating Src-family protein tyrosine kinase activation.
J. Immunol.
162
:
7088
7094
.
15
Fujimoto
M.
,
Fujimoto
Y.
,
Poe
J. C.
,
Jansen
P. J.
,
Lowell
C. A.
,
DeFranco
A. L.
,
Tedder
T. F.
.
2000
.
CD19 regulates Src family protein tyrosine kinase activation in B lymphocytes through processive amplification.
Immunity
13
:
47
57
.
16
Fearon
D. T.
,
Carroll
M. C.
.
2000
.
Regulation of B lymphocyte responses to foreign and self-antigens by the CD19/CD21 complex.
Annu. Rev. Immunol.
18
:
393
422
.
17
Carter
R. H.
,
Barrington
R. A.
.
2004
.
Signaling by the CD19/CD21 complex on B cells.
Curr. Dir. Autoimmun.
7
:
4
32
.
18
Del Nagro
C. J.
,
Otero
D. C.
,
Anzelon
A. N.
,
Omori
S. A.
,
Kolla
R. V.
,
Rickert
R. C.
.
2005
.
CD19 function in central and peripheral B-cell development.
Immunol. Res.
31
:
119
131
.
19
Tedder
T. F.
,
Poe
J. C.
,
Fujimoto
M.
,
Haas
K. M.
,
Sato
S.
.
2005
.
The CD19-CD21 signal transduction complex of B lymphocytes regulates the balance between health and autoimmune disease: systemic sclerosis as a model system.
Curr. Dir. Autoimmun.
8
:
55
90
.
20
Rickert
R. C.
,
Rajewsky
K.
,
Roes
J.
.
1995
.
Impairment of T-cell-dependent B-cell responses and B-1 cell development in CD19-deficient mice.
Nature
376
:
352
355
.
21
Engel
P.
,
Zhou
L. J.
,
Ord
D. C.
,
Sato
S.
,
Koller
B.
,
Tedder
T. F.
.
1995
.
Abnormal B lymphocyte development, activation, and differentiation in mice that lack or overexpress the CD19 signal transduction molecule.
Immunity
3
:
39
50
.
22
Sato
S.
,
Ono
N.
,
Steeber
D. A.
,
Pisetsky
D. S.
,
Tedder
T. F.
.
1996
.
CD19 regulates B lymphocyte signaling thresholds critical for the development of B-1 lineage cells and autoimmunity.
J. Immunol.
157
:
4371
4378
.
23
Inaoki
M.
,
Sato
S.
,
Weintraub
B. C.
,
Goodnow
C. C.
,
Tedder
T. F.
.
1997
.
CD19-regulated signaling thresholds control peripheral tolerance and autoantibody production in B lymphocytes.
J. Exp. Med.
186
:
1923
1931
.
24
Fujimoto
M.
,
Sato
S.
.
2007
.
B cell signaling and autoimmune diseases: CD19/CD22 loop as a B cell signaling device to regulate the balance of autoimmunity.
J. Dermatol. Sci.
46
:
1
9
.
25
Theofilopoulos
A. N.
,
Dixon
F. J.
.
1985
.
Murine models of systemic lupus erythematosus.
Adv. Immunol.
37
:
269
390
.
26
Wither
J. E.
,
Roy
V.
,
Brennan
L. A.
.
2000
.
Activated B cells express increased levels of costimulatory molecules in young autoimmune NZB and (NZB x NZW)F(1) mice.
Clin. Immunol.
94
:
51
63
.
27
Shirai
T.
,
Hirose
S.
,
Okada
T.
,
Nishimura
H.
.
1991
.
CD5+ B cells in autoimmune disease and lymphoid malignancy.
Clin. Immunol. Immunopathol.
59
:
173
186
.
28
Hayakawa
K.
,
Hardy
R. R.
,
Parks
D. R.
,
Herzenberg
L. A.
.
1983
.
The “Ly-1 B” cell subpopulation in normal immunodefective, and autoimmune mice.
J. Exp. Med.
157
:
202
218
.
29
Atencio
S.
,
Amano
H.
,
Izui
S.
,
Kotzin
B. L.
.
2004
.
Separation of the New Zealand Black genetic contribution to lupus from New Zealand Black determined expansions of marginal zone B and B1a cells.
J. Immunol.
172
:
4159
4166
.
30
Mizoguchi
A.
,
Bhan
A. K.
.
2006
.
A case for regulatory B cells.
J. Immunol.
176
:
705
710
.
31
Mauri
C.
,
Ehrenstein
M. R.
.
2008
.
The ‘short’ history of regulatory B cells.
Trends Immunol.
29
:
34
40
.
32
Bouaziz
J. D.
,
Yanaba
K.
,
Tedder
T. F.
.
2008
.
Regulatory B cells as inhibitors of immune responses and inflammation.
Immunol. Rev.
224
:
201
214
.
33
Fillatreau
S.
,
Gray
D.
,
Anderton
S. M.
.
2008
.
Not always the bad guys: B cells as regulators of autoimmune pathology.
Nat. Rev. Immunol.
8
:
391
397
.
34
Yanaba
K.
,
Bouaziz
J. D.
,
Haas
K. M.
,
Poe
J. C.
,
Fujimoto
M.
,
Tedder
T. F.
.
2008
.
A regulatory B cell subset with a unique CD1dhiCD5+ phenotype controls T cell-dependent inflammatory responses.
Immunity
28
:
639
650
.
35
Wolf
S. D.
,
Dittel
B. N.
,
Hardardottir
F.
,
Janeway
C. A.
 Jr.
.
1996
.
Experimental autoimmune encephalomyelitis induction in genetically B cell-deficient mice.
J. Exp. Med.
184
:
2271
2278
.
36
Mizoguchi
A.
,
Mizoguchi
E.
,
Smith
R. N.
,
Preffer
F. I.
,
Bhan
A. K.
.
1997
.
Suppressive role of B cells in chronic colitis of T cell receptor alpha mutant mice.
J. Exp. Med.
186
:
1749
1756
.
37
Mizoguchi
A.
,
Mizoguchi
E.
,
Takedatsu
H.
,
Blumberg
R. S.
,
Bhan
A. K.
.
2002
.
Chronic intestinal inflammatory condition generates IL-10-producing regulatory B cell subset characterized by CD1d upregulation.
Immunity
16
:
219
230
.
38
Fillatreau
S.
,
Sweenie
C. H.
,
McGeachy
M. J.
,
Gray
D.
,
Anderton
S. M.
.
2002
.
B cells regulate autoimmunity by provision of IL-10.
Nat. Immunol.
3
:
944
950
.
39
Mauri
C.
,
Gray
D.
,
Mushtaq
N.
,
Londei
M.
.
2003
.
Prevention of arthritis by interleukin 10-producing B cells.
J. Exp. Med.
197
:
489
501
.
40
Matsushita
T.
,
Yanaba
K.
,
Bouaziz
J. D.
,
Fujimoto
M.
,
Tedder
T. F.
.
2008
.
Regulatory B cells inhibit EAE initiation in mice while other B cells promote disease progression.
J. Clin. Invest.
118
:
3420
3430
.
41
Watanabe
R.
,
Fujimoto
M.
,
Ishiura
N.
,
Kuwano
Y.
,
Nakashima
H.
,
Yazawa
N.
,
Okochi
H.
,
Sato
S.
,
Tedder
T. F.
,
Tamaki
K.
.
2007
.
CD19 expression in B cells is important for suppression of contact hypersensitivity.
Am. J. Pathol.
171
:
560
570
.
42
Matsushita
T.
,
Fujimoto
M.
,
Hasegawa
M.
,
Komura
K.
,
Takehara
K.
,
Tedder
T. F.
,
Sato
S.
.
2006
.
Inhibitory role of CD19 in the progression of experimental autoimmune encephalomyelitis by regulating cytokine response.
Am. J. Pathol.
168
:
812
821
.
43
Passwell
J.
,
Schreiner
G. F.
,
Nonaka
M.
,
Beuscher
H. U.
,
Colten
H. R.
.
1988
.
Local extrahepatic expression of complement genes C3, factor B, C2, and C4 is increased in murine lupus nephritis.
J. Clin. Invest.
82
:
1676
1684
.
44
van Zelm
M. C.
,
Reisli
I.
,
van der Burg
M.
,
Castaño
D.
,
van Noesel
C. J.
,
van Tol
M. J.
,
Woellner
C.
,
Grimbacher
B.
,
Patiño
P. J.
,
van Dongen
J. J.
,
Franco
J. L.
.
2006
.
An antibody-deficiency syndrome due to mutations in the CD19 gene.
N. Engl. J. Med.
354
:
1901
1912
.
45
Moutsopoulos
H. M.
,
Boehm-Truitt
M.
,
Kassan
S. S.
,
Chused
T. M.
.
1977
.
Demonstration of activation of B lymphocytes in New Zealand black mice at birth by an immunoradiometric assay for murine IgM.
J. Immunol.
119
:
1639
1644
.
46
Cohen
P. L.
,
Ziff
M.
.
1977
.
Abnormal polyclonal B cell activation in NZB/NZW F1 mice.
J. Immunol.
119
:
1534
1537
.
47
Izui
S.
,
McConahey
P. J.
,
Dixon
F. J.
.
1978
.
Increased spontaneous polyclonal activation of B lymphocytes in mice with spontaneous autoimmune disease.
J. Immunol.
121
:
2213
2219
.
48
Klinman
D. M.
1990
.
Polyclonal B cell activation in lupus-prone mice precedes and predicts the development of autoimmune disease.
J. Clin. Invest.
86
:
1249
1254
.
49
Fujimoto
M.
,
Poe
J. C.
,
Satterthwaite
A. B.
,
Wahl
M. I.
,
Witte
O. N.
,
Tedder
T. F.
.
2002
.
Complementary roles for CD19 and Bruton’s tyrosine kinase in B lymphocyte signal transduction.
J. Immunol.
168
:
5465
5476
.
50
Buhl
A. M.
,
Cambier
J. C.
.
1999
.
Phosphorylation of CD19 Y484 and Y515, and linked activation of phosphatidylinositol 3-kinase, are required for B cell antigen receptor-mediated activation of Bruton’s tyrosine kinase.
J. Immunol.
162
:
4438
4446
.
51
Yanaba
K.
,
Bouaziz
J. D.
,
Matsushita
T.
,
Tsubata
T.
,
Tedder
T. F.
.
2009
.
The development and function of regulatory B cells expressing IL-10 (B10 cells) requires antigen receptor diversity and TLR signals.
J. Immunol.
182
:
7459
7472
.
52
Abe
J.
,
Ueha
S.
,
Suzuki
J.
,
Tokano
Y.
,
Matsushima
K.
,
Ishikawa
S.
.
2008
.
Increased Foxp3(+) CD4(+) regulatory T cells with intact suppressive activity but altered cellular localization in murine lupus.
Am. J. Pathol.
173
:
1682
1692
.
53
Moore
K. W.
,
de Waal Malefyt
R.
,
Coffman
R. L.
,
O’Garra
A.
.
2001
.
Interleukin-10 and the interleukin-10 receptor.
Annu. Rev. Immunol.
19
:
683
765
.
54
Haas
K. M.
,
Watanabe
R.
,
Matsushita
T.
,
Nakashima
H.
,
Ishiura
N.
,
Okochi
H.
,
Fujimoto
M.
,
Tedder
T. F.
.
2010
.
Protective and pathogenic roles for B cells during systemic autoimmunity in NZB/W F1 mice.
J. Immunol.
184
:
4789
4800
.
55
Shi
X.
,
Xie
C.
,
Chang
S.
,
Zhou
X. J.
,
Tedder
T.
,
Mohan
C.
.
2007
.
CD19 hyperexpression augments Sle1-induced humoral autoimmunity but not clinical nephritis.
Arthritis Rheum.
56
:
3057
3069
.
56
Chan
O. T.
,
Hannum
L. G.
,
Haberman
A. M.
,
Madaio
M. P.
,
Shlomchik
M. J.
.
1999
.
A novel mouse with B cells but lacking serum antibody reveals an antibody-independent role for B cells in murine lupus.
J. Exp. Med.
189
:
1639
1648
.
57
Waters
S. T.
,
McDuffie
M.
,
Bagavant
H.
,
Deshmukh
U. S.
,
Gaskin
F.
,
Jiang
C.
,
Tung
K. S.
,
Fu
S. M.
.
2004
.
Breaking tolerance to double stranded DNA, nucleosome, and other nuclear antigens is not required for the pathogenesis of lupus glomerulonephritis.
J. Exp. Med.
199
:
255
264
.
58
Christensen
S. R.
,
Kashgarian
M.
,
Alexopoulou
L.
,
Flavell
R. A.
,
Akira
S.
,
Shlomchik
M. J.
.
2005
.
Toll-like receptor 9 controls anti-DNA autoantibody production in murine lupus.
J. Exp. Med.
202
:
321
331
.
59
Mosser
D. M.
,
Zhang
X.
.
2008
.
Interleukin-10: new perspectives on an old cytokine.
Immunol. Rev.
226
:
205
218
.
60
Houssiau
F. A.
,
Lefebvre
C.
,
Vanden Berghe
M.
,
Lambert
M.
,
Devogelaer
J. P.
,
Renauld
J. C.
.
1995
.
Serum interleukin 10 titers in systemic lupus erythematosus reflect disease activity.
Lupus
4
:
393
395
.
61
Llorente
L.
,
Zou
W.
,
Levy
Y.
,
Richaud-Patin
Y.
,
Wijdenes
J.
,
Alcocer-Varela
J.
,
Morel-Fourrier
B.
,
Brouet
J. C.
,
Alarcon-Segovia
D.
,
Galanaud
P.
,
Emilie
D.
.
1995
.
Role of interleukin 10 in the B lymphocyte hyperactivity and autoantibody production of human systemic lupus erythematosus.
J. Exp. Med.
181
:
839
844
.
62
Hagiwara
E.
,
Gourley
M. F.
,
Lee
S.
,
Klinman
D. K.
.
1996
.
Disease severity in patients with systemic lupus erythematosus correlates with an increased ratio of interleukin-10:interferon-gamma-secreting cells in the peripheral blood.
Arthritis Rheum.
39
:
379
385
.
63
Liu
T. F.
,
Jones
B. M.
.
1998
.
Impaired production of IL-12 in systemic lupus erythematosus. I. Excessive production of IL-10 suppresses production of IL-12 by monocytes.
Cytokine
10
:
140
147
.
64
González-Amaro
R.
,
Portales-Pérez
D.
,
Baranda
L.
,
Abud-Mendoza
C.
,
Llorente
L.
,
Richaud-Patin
Y.
,
Alcocer-Varela
J.
,
Alarcón-Segovia
D.
.
1998
.
Role of IL-10 in the abnormalities of early cell activation events of lymphocytes from patients with systemic lupus erythematosus.
J. Autoimmun.
11
:
395
402
.
65
Gröndal
G.
,
Gunnarsson
I.
,
Rönnelid
J.
,
Rogberg
S.
,
Klareskog
L.
,
Lundberg
I.
.
2000
.
Cytokine production, serum levels and disease activity in systemic lupus erythematosus.
Clin. Exp. Rheumatol.
18
:
565
570
.
66
Chun
H. Y.
,
Chung
J. W.
,
Kim
H. A.
,
Yun
J. M.
,
Jeon
J. Y.
,
Ye
Y. M.
,
Kim
S. H.
,
Park
H. S.
,
Suh
C. H.
.
2007
.
Cytokine IL-6 and IL-10 as biomarkers in systemic lupus erythematosus.
J. Clin. Immunol.
27
:
461
466
.
67
Yin
Z.
,
Bahtiyar
G.
,
Zhang
N.
,
Liu
L.
,
Zhu
P.
,
Robert
M. E.
,
McNiff
J.
,
Madaio
M. P.
,
Craft
J.
.
2002
.
IL-10 regulates murine lupus.
J. Immunol.
169
:
2148
2155
.
68
Ishida
H.
,
Muchamuel
T.
,
Sakaguchi
S.
,
Andrade
S.
,
Menon
S.
,
Howard
M.
.
1994
.
Continuous administration of anti-interleukin 10 antibodies delays onset of autoimmunity in NZB/W F1 mice.
J. Exp. Med.
179
:
305
310
.
69
Martin
F.
,
Chan
A. C.
.
2006
.
B cell immunobiology in disease: evolving concepts from the clinic.
Annu. Rev. Immunol.
24
:
467
496
.
70
Chan
O.
,
Shlomchik
M. J.
.
1998
.
A new role for B cells in systemic autoimmunity: B cells promote spontaneous T cell activation in MRL-lpr/lpr mice.
J. Immunol.
160
:
51
59
.
71
Li
Y.
,
Chen
F.
,
Putt
M.
,
Koo
Y. K.
,
Madaio
M.
,
Cambier
J. C.
,
Cohen
P. L.
,
Eisenberg
R. A.
.
2008
.
B cell depletion with anti-CD79 mAbs ameliorates autoimmune disease in MRL/lpr mice.
J. Immunol.
181
:
2961
2972
.
72
Martin
F.
,
Chan
A. C.
.
2004
.
Pathogenic roles of B cells in human autoimmunity; insights from the clinic.
Immunity
20
:
517
527
.