Self DNA from Lymphocytes That Have Undergone Activation-Induced Cell Death Enhances Murine B Cell Proliferation and Antibody Production

<div><p>Systemic lupus erythematosus (SLE) is characterized by prominent autoinflammatory tissue damage associated with impaired removal of dying cells and DNA. Self DNA-containing immune complexes are able to activate both innate and adaptive immune responses and play an important role in the maintenance and exacerbation of autoimmunity in SLE. In this study, we used DNA from lymphocytes that have undergone activation-induced cell death (ALD-DNA) and analyzed its role on the activation and differentiation of B cells from normal BALB/c mice as well as lupus-prone MRL^+/+ and MRL/lpr mice. We found that ALD-DNA directly increased the expression of costimulatory molecules and the survival of naïve B cells <i>in vitro</i>. Although ALD-DNA alone had little effect on the proliferation of naïve B cells, it enhanced LPS-activated B cell proliferation <i>in vitro</i> and <i>in vivo</i>. In addition, ALD-DNA increased plasma cell numbers and IgG production in LPS-stimulated cultures of naïve B cells, in part via enhancing IL-6 production. Importantly, B cells from lupus mice were hyperresponsive to ALD-DNA and/or LPS relative to normal control B cells in terminal plasma cell differentiation, as evidenced by increases in CD138⁺ cell numbers, IgM production, and mRNA levels of B lymphocyte-induced maturation protein-1 (Blimp-1) and the X-box binding protein 1 (XBP1). Furthermore, ALD-DNA enhanced CD40-activated naïve B cell proliferation. Collectively, these data indicate that self DNA can serve as a DAMP (damage-associated molecular pattern) that cooperates with signals from both innate and adaptive immunity to promote polyclonal B cell activation, a common characteristic of autoimmune diseases.</p></div

Enhancement of CD40-stimulated naïve B cell proliferation by ALD-DNA.

<p>(<b>A</b>) Naïve B cells were pre-incubated for 20 min with polymyxin B (PMB; 20 µg/ml) before the addition of ALD-DNA (50 µg/ml), LPS (100 ng/ml), anti-CD40 (1 µg/ml), LPS (100 ng/ml) plus ALD-DNA (50 µg/ml), or anti-CD40 (1 µg/ml) plus ALD-DNA (50 µg/ml). After 72 h stimulation, cell proliferation was detected by performing BrdU incorporation assay using ELISA. Data are presented as the means ±SEM of three independent experiments. (<b>B</b>) CFSE-labeled naïve B cells were cultured in media containing ALD-DNA, anti-CD40, or both (at the concentrations as described above) for 72 h. Cell division was monitored by measuring the dilution of CFSE. Shaded areas show the cell division peaks predicted by the ModFit software. Results from one representative experiment of three are shown. **<i>P</i><0.01.</p

Effect of ALD-DNA on CD138, Blimp-1, XBP1, and IL-6 expression in naïve B cells.

<p>Naïve B cells were cultured in media containing ALD-DNA (50 µg/ml), LPS (100 ng/ml), or both for different periods as described below. (<b>A</b>) After 72 h stimulation, Cells were analyzed for B220 and CD138 surface expression by flow cytometry. The number indicates the percentage of B220⁻CD138⁺ cells in the gate. Similar results were obtained in three independent experiments. (<b>B</b>) Expression of Blimp-1 and XBP1 mRNA from B cells cultured for 96 hours was examined by real-time PCR. Fold induction relative to their untreated controls is shown (means ±SEM, n = 5). (<b>C</b>) Expression of IL-6 mRNA in B cells after 6 h stimulation was examined by real-time PCR. Fold induction relative to their untreated controls is shown (means ±SEM, n = 5); The amounts of IL-6 protein in the culture supernatants of B cells after 72 h stimulation were measured by ELISA (n = 6, a line linked two dots represents a pair observation). Up-regulation of IL-6 protein was undetectable in all samples after ALD-DNA treatment. *<i>P</i><0.05 as compared to the LPS, ***<i>P</i><0.001 as compared to the LPS.</p

Synergistic enhancement of LPS-driven IgG production in naive B cells by ALD-DNA stimulation.

<p>Naive B cells were cultured for 6 days in media containing ALD-DNA (50 µg/ml) with or without LPS (100 ng/ml). Cell culture supernatants were collected, and the levels of IgM and IgG were measured by ELISA. Bars represent the means ±SEM of three independent experiments (n = 5). ND (not detected). **<i>P</i><0.01.</p

Up-regulation of CD86 and MHC class II expression in naïve B cells by ALD-DNA stimulation.

<p>(<b>A</b>) Naïve B cells were incubated with ALD-DNA (50 µg/ml) in the presence or absence of LPS ranging from 0.01 µg/mL to 10 µg/ml for 72 h. The numbers of CD86-expressing B cells after each treatment were determined by flow cytometry. (<b>B</b>) Naive B cells were stimulated with ALD-DNA or <i>E. coli</i> single-stranded (ss) DNA (both at 50 µg/ml) for 72 h. CD86 and MHC class II expression was determined by performing flow cytometry. Histogram plots show the mean fluorescence intensity (MFI) of these surface molecules. The data are representative of three independent experiments.</p

Promotion of plasma cell differentiation and Ig production in lupus B cells by ALD-DNA.

<p>Splenic B cells from 16- to 20-week-old MRL/lpr mice and 12-week-old MRL^+/+ mice were cultured in media alone, or in the presence of ALD-DNA (50 µg/ml), LPS (100 ng/ml), or both for different periods as indicated below. (<b>A</b>) After 72 h stimulation, viable cells were analyzed for B220 and CD138 surface expression by flow cytometry. Representative dot plots showed the frequencies of plasmablasts (B220⁺CD138^hi, lower panels, MRL/lpr, n = 6) or plasma cells (B220⁻CD138⁺, upper panels, MRL^+/+, n = 6) generated after each treatment. (<b>B</b>) Expression of Blimp-1 and XBP1 mRNA from lupus B cells cultured for 96 h was examined by real-time PCR. Results are expressed as the fold induction of gene transcription relative to their untreated controls (means ±SEM, n = 6). (<b>C</b>) Ig production in cell culture supernatants harvested on day 6 was determined by ELISA. Bars represent the means ±SEM of three independent experiments (n = 6). *<i>P</i><0.05, **<i>P</i><0.01.</p

IgM from BALB/c mice enhances germinal center responses.

<p>On day 0, BALB/c mice were immunized i.v. with WT or Cµ13 IgM specific for KLH (50 µg/mouse) or SRBC (0.2 ml of a solution with HA titer 1:32) 30 min before 10 µg KLH (A), 5×10⁵ (B) or 5×10⁶ SRBC (C) were administered via the same route; controls received antigens or specific IgM alone. Spleens were harvested on day 10. Splenocytes from half of each spleen were analyzed by flow cytometry; germinal center B cells were gated as GL7⁺PNA⁺ amongst B220⁺ cells (Figure S1) and the percentages of germinal center B cells were quantified (A-C, upper left panels). The other halves of the spleens were sectioned, stained with anti-B220 (blue), anti-MOMA (green) and PNA (red), and analyzed for number of PNA⁺ germinal centers in B cell follicles by confocal microscopy (A-C, upper right panels); each image is a representative area (1725 µm × 1295 µm) for 2-3 whole sections with original magnification ×10 (A-C, lower panels). Germinal center responses of mice immunized with specific IgM alone were always lower than the responses of mice immunized with antigens alone (not shown). Data are representative of two experiments with each antigen dose. ns = not significant; * = p < 0.05; ** = p < 0.01.</p

IgM from Cμ13 mice cannot enhance antibody responses.

<p>BALB/c mice were immunized i.v. with WT or Cµ13 IgM anti-KLH (50 µg/mouse) or anti-SRBC (0.2 ml of a solution with HA titer 1:8). Thirty minutes later, 10 µg KLH, 5×10⁵ or 5×10⁶ SRBC were administered. Mice immunized with only antigen or IgM were used as controls. All groups were bled at indicated time points and sera were screened for IgG anti-KLH (A) or IgG anti-SRBC (B, C). Antibody responses of mice immunized with specific IgM only were much lower than that of mice immunized with antigens alone (data not shown). Data represent three (A, B) or two (C) experiments. Statistical comparison is shown between mice given antigen alone and mice given antigen together with WT IgM (above) or Cµ13 IgM (below). ns = not significant; * = p < 0.05; ** = p < 0.01; *** = p < 0.001.</p

WT IgM induces rapid deposition of C3 on SRBC in the blood.

<p>BALB/c mice were immunized i.v. with IgM anti-SRBC (0.2 ml of a solution with HA titer 1:32) from WT BALB/c (WT IgM; n = 2) or Cµ13 mice (Cµ13 IgM; n = 2). Thirty minutes later, 5×10⁸ SRBC were administered. Mice given 5×10⁸ SRBC alone were used as controls (n=2). Peripheral blood samples were taken 1 min after immunization and immediately mixed with 1 µl lepirudin (50 mg/ml). Rabbit IgG anti-SRBC was used to identify SRBC in the blood (left panel). The deposition of C3 on SRBC in the blood from mice immunized with SRBC alone (black) or together with WT IgM (red) or Cµ13 IgM (blue) was analyzed (right panel). Representative of three independent experiments.</p

Activation of antigen-specific CD4⁺ T cells is not enhanced by specific IgM.

<p>BALB/c mice were adoptively transferred with 15×10⁶ splenocytes from DO11.10 mice. The next day, mice were immunized with 5×10⁶ SRBC-OVA alone or together with WT IgM anti-SRBC (0.2 ml of a solution with HA titer 1:8), 5×10⁸ SRBC-OVA (positive controls) or 5×10⁸ unconjugated SRBC (negative controls). One, 2, 3, and 4 days after immunization, spleens from 2-3 mice per group were harvested and analyzed by flow cytometry. Kinetics of KJ1-26⁺CD4⁺ cell expansion (A), percentages of blasts among KJ1-26⁺CD4⁺ cells (B, left panel) and expression of surface markers LFA-1 (C, left panel) and CD44 (D, left panel) on KJ1-26⁺CD4⁺ cells were quantified. Numbers in histograms indicate the percentages of blasts, LFA-1^high and CD44^high cells among KJ1-26⁺CD4⁺ cells in the positive controls. Representative histograms (B-D, right panels) of each group immunized with 5×10⁸ SRBC-OVA (black), 5×10⁸ unconjugated-SRBC (grey), 5×10⁶ SRBC-OVA alone (blue) or together with WT IgM anti-SRBC (red) are shown for day 3. SEM were frequently too small to be seen in the figures.</p