RAGE-independent autoreactive B cell activation in response to chromatin and HMGB1/DNA immune complexes

Abstract

Increasing evidence suggests that the excessive accumulation of apoptotic or necrotic cellular debris may contribute to the pathology of systemic autoimmune disease. HMGB1 is a nuclear DNA-associated protein, which can be released from dying cells thereby triggering inflammatory processes. We have previously shown that IgG2a-reactive B cell receptor (BCR) transgenic AM14 B cells proliferate in response to endogenous chromatin immune complexes (ICs), in the form of the anti-nucleosome antibody PL2-3 and cell debris, in a TLR9-dependent manner, and that these ICs contain HMGB1. Activation of AM14 B cells by these chromatin ICs was inhibited by a soluble form of the HMGB1 receptor, RAGE-Fc, suggesting HMGB1-RAGE interaction was important for this response. To further explore the role of HMGB1 in autoreactive B cell activation, we assessed the capacity of purified calf thymus HMGB1 to bind dsDNA fragments and found that HMGB1 bound both CG-rich and CG-poor DNA. However, HMGB1-DNA complexes could not activate AM14 B cells unless HMGB1 was bound by IgG2a and thereby able to engage the BCR. To ascertain the role of RAGE in autoreactive B cell responses to chromatin ICs, we intercrossed AM14 and RAGE-deficient mice. We found that spontaneous and defined DNA ICs activated RAGE+ and RAGE(- ) AM14 B cells to a comparable extent. These results suggest that HMGB1 promotes B cell responses to endogenous TLR9 ligands through a RAGE-independent mechanism.

Figure 1. HMGB1 binds mammalian DNA in a sequence independent manner, and promotes TLR9-dependent responses when bound to CG-rich DNA and delivered through the BCR

(A) CG-rich (Clone 11 and CG50) and CG-poor (SUMO and CGneg) dsDNA at 800 ng/ml were incubated in the presence or absence of 10 μg/ml of HMGB1 for 1 h at 37°C, and ran in a agarose gel with Ethidium bromide. (B) 10 μg/ml of HMGB1 alone or in the presence of 250 ng/ml of unlabelled dsDNA fragments CG50 (G50) or CGneg (Gng) were added to AM14 B cells. HMGB1 alone or in the presence of CG50, CGneg, Clone 11 (C11) or Sumo (SU) were incubated before addition of 5 μg/ml of anti-HMGB1 IgG2a antibody. The resulting complexes were added to AM14 B cells and proliferation determined by [³H] thymidine incorporation. The TLR2 ligand Pam₄CysK₄ (P3Cysk) at 1 μg/ml was used as a control. (C) HMGB1 alone or in complex with anti-HMGB1, or CG50/HMGB1/anti-HMGB1 complexes were added to AM14 B cells from TLR9 sufficient (grey bars) or TLR9−/− mice (white bars). As controls, the TLR9 ligand 1826 (CpG) and TLR2 ligand Pam3CysK₄ (P3CysK), both at 1 μg/ml were added. In (A), shown is a representative experiment of at least three. (B) and (C), shown are the mean+SEM of three independent experiments.

Figure 2. HMGB1 does not enhance AM14 B cell proliferation by DNA-fragment ICs

(A) AM14 B cells were incubated with PL2-3 (left) or PA4 (right) in the presence (open squares) or absence (closed circles) of 1 μg/ml HMGB1. (B) AM14 B cells were incubated with the anti-DNA IgG2a antibody PA4, PA4/CG50, PA4/CGneg, anti-biotin IgG2a 1D4, 1D4/Bio-Clone 11, 1D4/Bio-Sumo in the absence (black bars) or the presence (open bars) of 1μg/ml HMGB1. As control, HMGB1 was incubated in the presence of the TLR2 ligand Pam3CysK₄. In (A) and (B), proliferation was determined as [³H] thymidine incorporation. Shown are a representative experiment (A) and mean+SEM of four experiments, except for 1D4/C11 and 1D4/SUMO, with mean+SEM of three (B).

Figure 3. RAGE deficiency does not affect B cell phenotype or responses to mitogens

(A) Splenic naïve B cells from RAGE+/− (RAGE⁺) and RAGE−/− (RAGE⁻) mice were stained with mouse anti-mouse RAGE antibody (left panel) or with mouse anti-Ig (right panel). (B and C) B220⁺ splenocytes from RAGE⁺ and RAGE⁻ mice were stained using anti-mouse CD93 (B) or anti-mouse CD21 and CD23 antibodies (C). Numbers represent percentages of each population. (D) Splenic naïve B cells from RAGE⁺ (grey bars) and RAGE⁻ (black bars) mice were incubated with 50 μg/ml anti-IgM F(ab)′₂, 0.1 μg/ml of the TLR9 ligand CpG ODN 1826 (CpG) and 30 ng/ml of the TLR7 ligand R848, and proliferation determined by [³H] thymidine incorporation. In (A–C), shown are representative results of three (A) and six (B and C) independent experiments. In (D), shown are mean+SEM of four (F(ab)′2), six (CpG) and three (R848) independent experiments.

Figure 4. RAGE-deficient AM14 B cells respond to chromatin ICs comparably to RAGE sufficient cells

(A and B) B220⁺ splenocytes from AM14 RAGE⁺ and RAGE⁻ mice were stained using anti-mouse CD93 (A), or anti-CD21 and CD23 antibodies (B) Numbers represent percentages of each population. (C) Splenic naïve B cells from RAGE⁺ (grey bars) and RAGE⁻ (black bars) mice were incubated with 0.03 μg/ml of PL2-3, 0.1 μg/ml PA4 plus 50 ng/ml CG50 ICs, 0.1 μg/ml CpG ODN 1826 (CpG) or 17 μg/ml anti-IgM (F(ab)′₂), and proliferation determined by [³H] thymidine incorporation. In (A and B), results are representative of six experiments. In (C), shown are the mean+SEM of seven (PL2-3), and five (PA4-CG50, F(ab)′₂, CpG) independent experiments.