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The Journal of Immunology, 2007, 179, 7397-7405
Copyright © 2007 by The American Association of Immunologists, Inc.
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Functional Outcome of B Cell Activation by Chromatin Immune Complex Engagement of the B Cell Receptor and TLR91

Liliana Busconi*, Jason W. Bauer{dagger}, Joseph R. Tumang{ddagger}, Amy Laws*, Kristin Perkins-Mesires*, Abigail S. Tabor*, Christina Lau*, Ronald B. Corley*, Thomas L. Rothstein{ddagger}, Frances E. Lund§, Timothy W. Behrens and Ann Marshak-Rothstein2,*

* Department of Microbiology, Boston University School of Medicine, Boston, MA 02118; {dagger} Department of Medicine, Center for Lupus Research, University of Minnesota School of Medicine, Minneapolis, MN 55455; {ddagger} Feinstein Institute for Medical Research, Manhasset, NY 11030; § Trudeau Institute, Saranac Lake, NY 12983; and Genentech, South San Francisco, CA 94080


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
We have previously shown that rheumatoid factors produced by Fas-deficient autoimmune-prone mice typically bind autologous IgG2a with remarkably low affinity. Nevertheless, B cells representative of this rheumatoid factor population proliferate vigorously in response to IgG2a/chromatin immune complexes through a mechanism dependent on the sequential engagement of the BCR and TLR9. To more precisely address the role of both receptors in this response, we analyzed the signaling pathways activated in AM14 B cells stimulated with these complexes. We found that the BCR not only serves to direct the chromatin complex to an internal compartment where it can engage TLR9 but also transmits a suboptimal signal that in combination with the signals emanating from TLR9 leads to NF-{kappa}B activation and proliferation. Importantly, engagement of both receptors leads to the up-regulation of a group of gene products, not induced by the BCR or TLR9 alone, that include IL-2. These data indicate that autoreactive B cells, stimulated by a combination of BCR and TLR9 ligands, acquire functional properties that may contribute to the activation of additional cells involved in the autoimmune disease process.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Despite the multitude of self-constituents that potentially serve as targets in systemic autoimmune diseases, the autoantibody repertoire is remarkably limited. In this context, DNA, chromatin-associated proteins, RNA, and ribonucleoprotein macromolecules stand out as particularly common autoantibody targets (1, 2). Very often, such anti-nuclear Abs are the first indication of autoimmune disease in both patients and in animal models of systemic lupus erythematosus. We have proposed that the frequent occurrence of anti-nuclear Abs is due to the capacity of nucleic acid-associated autoantigens to engage both the BCR and a member of the TLR gene family, either TLR9 or TLR7 (3, 4).

This activation process can be best examined in B cells representative of the actual autoantibody repertoire. Our laboratory has used the BCR-transgenic mouse line, AM14, to better understand the relationship between nucleic acid autoantigens and the development of autoimmune diseases. The AM14 receptor is a rheumatoid factor derived from an autoimmune Fas-deficient mouse that binds IgG2a with relatively low affinity (5). AM14 B cells proliferate robustly in response to IgG2a immune complexes (ICs)3 in which IgG2a is bound to chromatin (chromatin ICs), CpG-enriched dsDNA fragments (DNA ICs), RNA, or snRNP but no activation takes place when the IC contains IgG2a bound to a protein Ag (6).

The proliferative response to both chromatin ICs and DNA-fragment ICs is suppressed by agents that interfere with endosome acidification as well as by oligodeoxynucleotide (ODN) inhibitors of TLR9. Moreover, AM14 B cells that either fail to express the adaptor protein MyD88 or express the 3d mutation (7), and cannot signal through TLR9 (or TLR7), do not respond to chromatin ICs, even though their response to anti-IgM F(ab')2 remains comparable to nontransgenic B cells (3, 8). Collectively, these data indicate that the AM14 BCR recognizes IgG2a bound to chromatin and then shuttles these ICs to an endosomal or lysosomal compartment where components of the complex engage TLR9 (or TLR7). These studies have not addressed the effect of IC engagement on the various branches of the BCR-signaling cascade or the capacity of such signals to modulate the functional properties of TLR9-activated AM14 B cells.

To more precisely address the functional outcome of BCR/TLR9 coengagement on the activation of AM14 B cells, we asked whether chromatin ICs could elicit the expression of gene products not induced by either the BCR or TLR9 alone. One notable effect was a shift in the pattern of cytokine production. This observation led us to examine in more detail the signal transduction pathways activated by chromatin ICs in wild-type and TLR-deficient AM14 responder populations. The current studies demonstrate that BCR/TLR engagement results in unique functional properties. Moreover, the BCR plays an active role in signal transduction but TRL9 (or TLR7) is absolutely required to drive proliferation of autoreactive cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

AM14, AM14 MyD88–/–, AM14 TLR9–/–, and AM14 3d mice have been described previously (3, 8). MyD88–/– and TLR9–/– mice were provided by Dr. S. Akira (Research Institute for Microbial Diseases, Osaka University, Osaka, Japan) and the 3d (Unc93B–/–) were provided by Dr. B. Beutler (The Scripps Research Institute, La Jolla, CA). The microarray analysis used B cells from MRL/+ AM14 mice. Mice were bred and maintained in microisolator cages at the Boston University School of Medicine Laboratory Animal Science Center. Procedures were approved by the Institutional Animal Care and Use Committee of Boston University Medical Center.

B cell preparation and in vitro stimulation

Splenic B cells were isolated by positive selection with anti-B220-coupled magnetic beads (Miltenyi Biotec). B cells were also isolated by negative selection using a combination of anti-CD43-, anti-CD4-, and anti-CD8-coupled magnetic beads. For MAPK experiments, B cells were rested overnight at 37°C in culture medium before the addition of ligands. For all other experiments, the cells were stimulated immediately after isolation. Proliferation was quantitated as previously described (9). F(ab')2 goat anti-mouse IgM (Jackson ImmunoResearch Laboratories), CpG phosphorothioate ODN 1826, 5'-TCCATgACgTTCCTgACgTT-3', (Trilink BioTechnologies), R848 (InvivoGen) were used as ligands. Hybridoma cell lines producing monoclonal IgG2a Abs specific for nucleosomes (PL2–3) (10) and trinitrophenol (TNP) (Hy1.2) (11) were provided by Drs. M. Monestier (Temple University, Philadelphia, PA) and M. Shlomchik (Yale University, New Haven, CT), respectively. For proliferation assays, PL2–3 was added directly to AM14 B cells. For the signaling studies, PL2–3 was preincubated for 2 h at 37°C with spent culture fluid; these preformed chromatin ICs were then added to B cell cultures at concentration of 5 or 20 µg/ml PL2–3. DNA fragment ICs were prepared as described previously (9). Some experiments included the inhibitors cyclosporin A (Biomol International), U0126 (Calbiochem), cycloheximide or chloroquine (Sigma-Aldrich). All reagents were endotoxin-free (<0.1U/ml) as determined by the Limulus amebocyte lysate assay (BioWhittaker).

Microarray gene chip analysis

Cells (3 x 106) were stimulated with different ligands for 6 h. Total RNA was converted into biotin-labeled cRNA (MessageAmp aRNA kit; Ambion) and hybridized to murine U430_2.0 probe arrays (Affymetrix) The arrays were subjected to mask corrections (Genedata Expressionist), and all target hybridization intensities were scaled to 1500 arbitrary units using Microarray Suite 5.0 (Affymetrix). To identify genes that were differentially expressed between medium (MED) and experimental (EXP) cultures, we used linear fold change (FC) (mean EXP/mean MED). To filter out genes with very low expression, we discarded genes with an average signal intensity of <100 across all samples. Gene arrays were run on two samples from medium, anti-IgM F(ab')2, CpG, and PL2–3 treated AM-14 B cell cultures. A third array from each condition plus three samples from cultures treated with both anti-IgM F(ab')2 and CpG were run to provide triplicate data. To determine genes selectively up- or down-regulated in PL2–3, the following criteria were used: 1) PL2–3 > 2 FC vs medium, 2) PL2–3 > 1.5 FC vs anti-IgM F(ab')2, PL2–3 > 1.5 FC vs CpG, 3) anti-IgM F(ab')2 and CpG conditions within 3 FC of medium (within 1.5 FC for down-regulated genes). Genes that did not show similar trends in the third sample run for each condition were excluded. For visualization of the arrays, each expression value was divided by the mean of the corresponding media-treated samples. These ratios were transformed into log2 space, and subjected to centered-average linkage clustering using CLUSTER and visualized by TREEVIEW software (12). An account has been created in the Geo site (www.ncbi.nlm.nih.gov/geo GSE6674).

Cytokine assays

Cell culture supernatants were recovered 24 h after stimulation. Cytokine levels were measured using Luminex-based multiplex cytometric bead arrays (13) or the OptEIA ELISA kit (BD Biosciences).

Western blot analysis

A total of 1.5 x 106 cells were stimulated for varying periods of time and lysed in Laemmli buffer. The lysates were resolved on 10% (w/v) SDS-PAGE and electroblotted onto nitrocellulose membranes. The following Abs were used: anti-phospho-Syk (Tyr525/526), anti-phospho-Src family (Tyr416), anti-phospho-phospholipase C{gamma}2 (PLC{gamma}2) (Tyr1217), anti-phospho-p44/42 MAPK (Thr202/Tyr204), anti-phospho-stress-activated protein kinase/JNK (Thr183/Tyr185), anti-phospho-p38 MAPK (Thr180/Tyr182), anti-β-actin (Cell Signaling Technology), anti-tubulin and goat anti-mouse IgG-HRP (Sigma-Aldrich), anti-c-rel, anti-lamin B, goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology), anti-ERK1–2 and anti-phosphotyrosine (4G10) biotin-conjugated (Upstate Biotechnology/Cell Signaling Technology), donkey anti-goat IgG-HRP (Jackson ImmunoResearch Laboratories). The polyclonal antiserum specific for T-bet was provided by Dr. L. Glimcher (Harvard School of Public Health, Boston, MA). Blots were stripped using the Re-blot Western Blot Recycling kit (Chemicon International Inc) and reprobed up to four times.

Calcium flux

AM14 MyD88-deficient B cells were loaded with Indo-1AM (Molecular Probes) at 1 µM for 30 min at 37°C, washed, and resuspended to a final concentration of 5 x 106 cells/ml. After 20-min incubation at room temperature, cells were stimulated and intracellular Ca2+ was evaluated by measuring fluorescence at 405 and 485 nm after excitation at 355 nm with a Mo-Flo flow cytometer (DakoCytomation). Data analysis was performed using FlowJo software (Tree Star).

EMSA

AM14 B cells were stimulated for 24 h. The preparation of nuclear extracts, EMSA, and supershift analysis was done as described previously (14). Samples were electrophoresed in 4.5% polyacrylamide/TBE gel and analyzed by autoradiography. Abs to c-Rel, RelB, p50, p52, and p65 (Santa Cruz Biotechnology) were used in supershift analysis.

Nuclear translocation of c-Rel

A total of 5 x 106 AM14 or AM14 3d B cells were stimulated for 3.5 h with the specified ligands. Nuclear extracts were prepared as described previously (14). Samples were resolved on 10% (w/v) SDS-PAGE and electroblotted onto nitrocellulose membranes. Immunoblots were probed with anti c-rel and anti-lamin B Abs. In addition, AM14 or AM14 3d B cells were placed into 8-well CC2-treated chamber slides (Labtek) and stimulated with the specified ligands for 3–4 h at 37°C. Cells were washed, fixed with 3% paraformaldehyde, permeabilized for 30 min with 0.5% Triton X-100, and stained with TRITC-conjugated anti-c-Rel (Santa Cruz Biotechnology) for 1 h. The nuclei were stained with 4',6'-diamidino-2-phenylindole. Cells were imaged with a Zeiss Axiovert 200M inverted microscope equipped with the Apotome system and viewed with Axiovision software (bar 20 µm; Carl Zeiss).

I{kappa}B-{alpha} degradation

A total of 2.5 x 106 AM14 B cells were preincubated for 1 h with 50 µg/ml cycloheximide. B cells were stimulated with preformed chromatin ICs in the presence or absence of chloroquine (2 µg/ml) for varying periods of time and lysed in Laemmli buffer. Cell lysates were resolved on 10% (w/v) SDS-PAGE and electroblotted onto nitrocellulose membranes. Immunoblots were probed with anti-I{kappa}B-{alpha} and anti-{alpha}-tubulin Abs and analyzed by densitometry.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Chromatin IC activation of AM14 B cells leads to IL-2 production

Chromatin ICs engage both the BCR and TLR9. To determine whether cross-talk between the two pathways induces a program of gene expression unique to chromatin IC stimulation, we compared the overall gene expression profile of AM14 B cells stimulated for 6 h with PL2–3 (chromatin ICs), anti-IgM F(ab')2, CpG, and a combination of anti-IgM F(ab')2 plus CpG as an experimental condition presumably equivalent to full engagement of both the BCR and TLR9. In three independent experiments, chromatin ICs selectively increased the expression level of 44 transcripts and reduced the expression level of 65 genes by 1.5-fold or more as compared with cells treated with either anti-IgM F(ab')2 or CpG alone (Fig. 1). A subset of these genes was similarly regulated by the combination of anti-IgM F(ab')2 plus CpG. These data suggest that chromatin IC activation of AM14 B cells results in a distinct genetic program. For example, IL-2 expression increased only after stimulation with PL2–3 or a combination of anti-IgM F(ab')2 plus CpG (Fig. 1A).


Figure 1
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  		FIGURE 1. Chromatin IC activation of AM14 B cells induces a unique program of gene expression. AM14 B cells were stimulated for 6 h with 15 µg/ml anti-IgM F(ab')2, 1 µg/ml CpG, 5 µg/ml chromatin ICs (PL2–3), or a combination of anti-IgM F(ab')2 plus CpG. A and B, respectively, depict genes that were specifically up-regulated or down-regulated by PL2–3.

 
To confirm that the RNA data reflected actual cytokine production, culture supernatants collected 24 h after stimulation were assayed for IL-2. Consistent with the mRNA data, AM14 B cells, isolated by our standard positive selection procedure, produced IL-2 in response to PL2–3 and anti-IgM F(ab')2 plus CpG, but not in response to anti-IgM F(ab')2 or CpG alone (Fig. 2A). To prove that the IL-2 was B cell-derived, B cells isolated by a rigorous negative selection were also analyzed. The negatively selected population was completely unresponsive to Con A or anti-CD3 (Table I), and was therefore devoid of T cells, but still produced IL-2 in response to chromatin ICs as well as the combination of anti-IgM F(ab')2 plus CpG (Fig. 2A). Moreover, nontransgenic B cells still produced IL-2 in response to anti-IgM F(ab')2 plus CpG but did not produce IL-2 in response to PL2–3, further eliminating a non-B cell population as the source of IL-2 (Fig. 2B).


Figure 2
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  		FIGURE 2. Chromatin IC activation of AM14 B cells leads to IL-2 production. A, Positively and negatively selected AM14 B cells and, B, positively selected nontransgenic B cells, were stimulated with 15 µg/ml anti-IgM F(ab')2, 1 µg/ml CpG, 5 µg/ml chromatin ICs (PL2–3), or a combination of anti-IgM F(ab')2 plus CpG, and culture fluids were collected at 20–24 h. C, The experiment was repeated with positively selected AM14 B cells as described in A but cells were preincubated for 1 h with medium alone or with the inhibitor CsA (50 ng/ml) before ligand addition. Proliferation of AM14 B cells stimulated in the absence/presence of CsA was measured at 24 h by incorporation of [3H]thymidine (proliferation data minus CsA/proliferation data plus CsA: anti-IgM F(ab')2 (161561/83136), CpG (311881/295672), PL2–3 (416903/346252), and anti-IgM F(ab')2 plus CpG (401227/354124). D, The experiment was repeated with positively selected AM14 B cells as described in A but using AM14 and AM14 TLR9-deficient B cells. The data represent mean ± SEM of four different experiments.

 

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  		Table I. Proliferative responses of purified AM14 B cell populations

 
We next addressed the role of BCR and TLR9 in IL-2 secretion. As shown previously (9), cyclosporin A (CsA), an inhibitor of BCR signaling, blocks proliferation of AM14 B cells stimulated with anti-IgM F(ab')2 or PL2–3, but not CpG. CsA also blocked IL-2 production in AM14 B cells stimulated with PL2–3 or the combination of anti-IgM F(ab')2 plus CpG (Fig. 2C). PL2–3 also failed to stimulate IL-2 production in AM14 TLR9-deficient B cells (Fig. 2D). Collectively, these data demonstrate that IL-2 secretion depends on signals emanating from both the BCR and TLR9.

Chromatin IC activation of AM14 B cells does not induce expression of cytokines normally induced by CpG

B cell stimulation by CpG alone, (as well as by other TLR ligands) routinely leads to the production of TNF-{alpha}, IL-6, lL-10, and IL-12 (13, 15). Despite the documented requirement for TLR9 in the PL2–3 response, the microarray data indicated minimal production of these cytokines in PL2–3-stimulated B cells. This trend was confirmed by ELISA; both AM14 and nontransgenic B cells stimulated with CpG, or the combination of anti-IgM F(ab') 2 plus CpG, secreted high amounts of these cytokines, whereas these cytokines were not produced by PL2–3-stimulated cells (Fig. 3). These data demonstrate that the signaling cascades activated by chromatin ICs do not simply recapitulate the sum of optimal TLR9 and BCR ligands.


Figure 3
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  		FIGURE 3. Chromatin IC activation of AM14 B cells does not induce the cytokines normally produced in response to CpG. AM14 (left panel) or nontransgenic (right panel) B cells were stimulated with 15 µg/ml anti-IgM F(ab')2, 1 µg/ml CpG, 5 µg/ml chromatin ICs (PL2–3), or a combination of anti-IgM F(ab')2 plus CpG. Culture fluids were collected at 20–24 h. The data in A represent mean ± SEM of four different experiments.

 
Chromatin ICs induce T-bet expression

One possible explanation for the inability of PL2–3 to induce these cytokines is that mammalian DNA ligands are not able to fully activate the TLR9-signaling cascades. To examine signaling events induced downstream of TLR9, we monitored the expression of T-bet, a member of the T-box family of transcription factors that is induced in B cells by engagement of TLR9, but not TLR4 or the BCR. Consistent with published reports (16), CpG, but not anti-IgM F(ab')2, markedly augmented T-bet expression, as determined by Western blot analysis of cell lysates collected at 20 h postactivation. Moreover, both chromatin ICs and dsDNA fragment ICs induced high levels of T-bet expression (Fig. 4). dsDNA ICs consisted of a 600-bp fragment of haptenated CpG-rich dsDNA bound the anti-TNP IgG2a mAb Hy1.2 (9). These dsDNA fragment ICs effectively stimulate AM14 B cell proliferation even though they bind less avidly to AM14 B cells than chromatin ICs (9).


Figure 4
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  		FIGURE 4. Chromatin ICs induce T-bet. B cells isolated from AM14 mice and AM14 TLR9–/– were stimulated with 15 µg/ml anti-IgM F(ab')2, 3 µg/ml CpG, 5 µg/ml PL2–3 (chromatin ICs), DNA ICs (25 µg/ml Hy1.2, 250 ng/ml CG50-TNP2), or 150 ng/ml R848 for 20 h. The responses were analyzed by Western blot of cell lysates using a polyclonal T-bet-specific antiserum. The blot was stripped and reprobed with anti-β-actin as a loading control.

 
T-bet induction by PL2–3 depended on the presence of an active TLR9-signaling pathway, as it was markedly reduced in AM14 B cells obtained from TLR9-deficient AM14 mice. T-bet expression could still be induced in TLR9-deficient cells by R848, a synthetic TLR7 ligand. These data demonstrate that mammalian DNA contained in the chromatin ICs was able to up-regulate T-bet through effective engagement of TLR9.

Chromatin IC-induced tyrosine phosphorylation does not require activation of TLRs

In response to the more common polyclonal BCR ligand, anti-IgM F(ab')2, the BCR transduces a signal that leads to the rapid tyrosine phosphorylation of intracellular molecules such as Src family kinases, Syk and PLC{gamma}2 (17), and the proliferation of both wild-type and MyD88-deficient B cells. To further examine the role of the BCR in chromatin IC-mediated activation of the AM14 receptor, we used AM14 MyD88-deficient B cells. These cells allowed us to analyze BCR signaling in the absence of an active TLR9-signaling cascade. Chromatin ICs were formed by premixing the mAb PL2–3 with spent culture fluid and initially used at a concentration of 5 µg/ml PL2–3. This concentration of PL2–3 routinely elicited the maximum level of proliferation of AM14 MyD88-sufficient B cells but fails to induce proliferation in AM14 MyD88-defficient cells (Fig. 5A).


Figure 5
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  		FIGURE 5. Chromatin IC-induced tyrosine phosphorylation does not require activation of TLRs. A, Proliferation of AM14 MyD88-sufficient and AM14 MyD88–/–-deficient B cells stimulated with increasing concentrations of PL2–3. Data are representative of three experiments with similar results. B, Upper panel, B cells from AM14 MyD88-deficient mice were stimulated with 15 µg/ml anti-IgM F(ab')2, 3 µg/ml CpG, or two concentrations of preformed chromatin ICs (5 and 20 µg/ml) for the indicated periods of time. The lysates were electrophoresed in SDS-PAGE and immunoblotted with anti phosphotyrosine (4G10). B, lower panel, Replicate membranes were probed using Abs against p-Syk, p-Src family protein tyrosine kinase, p-PLC{gamma}2, and β-actin (loading control). Similar data were obtained in two additional experiments.

 
Stimulation of AM14 MyD88-deficient B cells with chromatin ICs induced a marked increase in overall protein tyrosine phosphorylation, as determined by Western blot using anti phospho-Tyr Ab (Fig. 5B, upper panel). To examine specific proteins, replicate membranes were probed with Abs against p-Syk, p-Src family members, and p-PLC{gamma}2 (Fig. 5B, lower panel). Chromatin ICs at 5 µg/ml, similarly to anti-IgM F(ab')2, induced a rapid activation of the Src protein tyrosine kinase family, but elicited very weak Syk phosphorylation and less intense and delayed PLC{gamma}2 activation. To determine whether this relatively weak response could be enhanced by more extensive cross-linking of the BCR, we stimulated AM14 B cells with a 4-fold higher concentration of chromatin ICs. Chromatin ICs at 20 µg/ml augmented Syk phosphorylation and accelerated the kinetics of PLC{gamma}2 activation. However, the proliferative response of MyD88-sufficient AM14 B cells to both 5 and 20 µg/ml PL2–3 was comparable and neither concentration was able to drive the proliferation of AM14 MyD88-deficient cells (3). Overall, these results support the notion that chromatin ICs suboptimally trigger components of the BCR signaling cascade in the absence of TLR engagement.

Chromatin ICs induce a diminished calcium response

Intracellular Ca2+ is a key second messenger in B cells following BCR engagement. We therefore examined calcium mobilization after chromatin IC stimulation of AM14 MyD88-deficient B cells. As observed for tyrosine phosphorylation, 5 µg/ml PL2–3 evoked less efficient response than cells stimulated with chromatin ICs at 20 µg/ml (Fig. 6, upper panel). Although chromatin ICs at 20 µg/ml triggered a greater level of calcium flux with faster kinetics, the levels of intracellular calcium rapidly decayed to that elicited by the lower chromatin IC concentration. Stimulation with anti-IgM F(ab')2 induced a rapid surge and a more sustained elevation of Ca2+ (Fig. 6, upper panel). As expected, CpG failed to trigger Ca2+ flux even in MyD88-sufficient cells (Fig. 6, lower panel). Taken together, these results indicate that AM14 B cells are not anergic and respond normally to anti-IgM F(ab')2 but that chromatin ICs induce suboptimal engagement of the BCR.


Figure 6
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  		FIGURE 6. Chromatin ICs induce a weak calcium response. AM14 MyD88-deficient B cells (upper panel) or wild-type (WT) MyD88-sufficient B cells (lower panel) were loaded with Ca2+-sensitive Indo-1AM. Cells were stimulated with the indicated ligands. Ca2+ was evaluated by measuring fluorescence at 405 and 485 nm after excitation at 355 nm with a Mo-Flo flow cytometer. The experiment shown is representative of four experiments with similar results.

 
Chromatin ICs activate MAPKs through engagement of BCR and TLR9

To further characterize the signaling pathways activated by chromatin ICs, we evaluated chromatin IC-induced MAPK activation. As previously reported for B cell lines (18), stimulation of AM14 B cells with anti-IgM F(ab')2, but not with CpG, led to rapid and sustained ERK activation (Fig. 7A). Both ligands induced phosphorylation of JNK and p38, but they elicited different overall phosphorylation patterns and distinct activation kinetics. Three phosphorylated isoforms of JNK were detected in extracts from anti-IgM F(ab')2-treated cells. All three bands detected by the anti p-JNK Ab corresponded to JNK isoforms as their phosphorylation could be blocked by the JNK inhibitor SP600125 (data not shown). Although the fastest migrating form was not detected after CpG treatment, chromatin IC stimulation induced phosphorylation of all three MAPK family members, further indicating a contribution of the BCR to the signaling response.


Figure 7
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  		FIGURE 7. Chromatin ICs activates different MAPKs. A, AM14 B cells were stimulated for different periods of time with 15 µg/ml anti-IgM F(ab')2, 3 µg/ml CpG, 5 µg/ml preformed chromatin ICs, or DNA ICs (25 µg/ml Hy1.2, 250 ng/ml CG50-TNP2). The responses were analyzed by Western blot using anti-p-ERK, anti-p-JNK, anti p-p38, and anti-ERK (loading control). Data are representative of more than four experiments that showed similar results. B, B cells isolated from AM14, AM14 MyD88–/–, and AM14 TLR9–/– mice were stimulated with anti-IgM F(ab')2, CpG, or preformed chromatin ICs for different periods of time and analyzed by Western blot using anti p-ERK. Anti-ERK was used as a loading control. C, AM14 B cells were preincubated for 1 h in the presence of increasing concentrations of U0126 and then stimulated with anti-IgM F(ab')2 ({circ}), CpG ({diamond}), or PL2–3 (chromatin ICs) ({square}). Proliferation was measured at 24 h by [3H]thymidine incorporation. Similar results were obtained in three experiments with similar results.

 
We also stimulated AM14 B cells with the dsDNA fragment ICs. The JNK phosphorylation pattern elicited by TNP2-CG50 ICs was very similar to that of the CpG-stimulated cells, although ERK phosphorylation was lower than in the chromatin ICs-stimulated cells (Fig. 7A). These results indicate that both chromatin ICs and the dsDNA fragment ICs activate MAPKs through both the BCR and TLR9, but that the contribution of the BCR can vary depending on the valency of the Ag.

Although CpG did not induce ERK phosphorylation in B cells, the ERK cascade can be activated by TLR9 ligands in other cell types (18, 19). To formally rule out a role of TLR9 in the ERK pathway of PL2–3-stimulated cells, we examined AM14 MyD88-deficient and AM14 TLR9-deficient cells. The pattern of ERK phosphorylation was similar in AM14 wild-type, MyD88–/–, and TLR9–/– cells, confirming that TLR9 did not contribute to the observed ERK activation (Fig. 7B). To address the contribution of ERK to the chromatin IC-induced proliferative response, we added U0216, an inhibitor of MEK, the kinase upstream of ERK, to the cultures. U0126 markedly reduced the activation of AM14 B cells stimulated with anti-IgM F(ab')2 and chromatin ICs but, as expected, had no effect on the response to CpG (Fig. 7C). This result further supports a significant contribution of the BCR to the chromatin IC-induced proliferative response.

Chromatin ICs require an active TLR9 to induce NF-{kappa}B activation

NF-{kappa}B plays an important role in the proliferative response of B cells following either BCR (anti-IgM F(ab')2) or TLR9 (CpG) engagement. As expected, anti-IgM F(ab')2, CpG, and PL2–3 all induced NF-{kappa}B activation in AM14 B cells, as shown by the ability of nuclear extracts to bind NF-{kappa}B target sequences (Fig. 8A). Ab supershift analysis revealed that all three treatments induced NF-{kappa}B dimers containing c-rel and/or p-50 (Fig. 8A) (20). To further examine the role of the BCR and TLR9 in AM14 B cell activation, we examined c-rel translocation to the nucleus in response to anti-IgM F(ab')2, CpG, or PL2–3 in AM14 wild-type and AM14 3d mutant cells (Fig. 8B). 3d AM14 B cells fail to signal through TLR9, TLR7, or TLR3 (7). In all cases, stimulation with anti-IgM F(ab')2 induced c-rel translocation to the nucleus. In contrast, CpG and PL2–3 induced c-rel translocation in WT AM14 cells but not in 3d AM14 B cells. Similar results were obtained by assessing c-rel translocation in AM14 and AM14 3d B cells by immunofluorescence (Fig. 8C). We confirmed the previous results by analyzing I{kappa}B degradation in wild-type AM14 B cells stimulated with PL2–3 in the presence or absence of chloroquine, a reagent known to increase the endosomal pH and specifically block TLR3/7/9 signaling (Fig. 8D). These results indicate that TLR9 is essential for NF-{kappa}B activation.


Figure 8
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  		FIGURE 8. Full activation of NF-{kappa}B requires engagement of TLR9. A, AM14 B cell were treated for 24 h with 15 µg/ml anti-IgM F(ab')2, 3 µg/ml CpG, 5 µg/ml PL2–3. Nuclear extracts were prepared and subjected to EMSA analysis. Samples from cells stimulated with PL2–3 were run in two separate gels (as indicated by the vertical line) under the same conditions. The identity of the NF-{kappa}B-binding proteins was evaluated by including Abs to p-50, p-52, p-65, c-Rel, and RelB in the binding reactions to supershift nucleoprotein complexes. The experiment was repeated twice with similar results. B, AM14 B cells prepared from wild-type (WT) mice or carrying the mutation 3d were treated for 3.5 h with medium, 15 µg/ml anti-IgM F(ab')2, 3 µg/ml CpG, and 5 and 20 µg/ml PL2–3. Nuclear extracts were prepared and analyzed by Western blot using a c-rel Ab. Equal protein loading was controlled by stripping the membrane and probing with anti-lamin B. c-rel translocation to the nucleus was quantified. The data represent mean ± SEM of three independent experiments. Arbitrary units: The c-Rel-lamin B ratio after stimulation with anti-IgM F(ab')2 was used as an internal control in each individual experiment and was considered equal to 100. The c-Rel-lamin B ratio for each ligand was expressed relative to the response to anti-IgM F(ab')2. C, B cells prepared from AM14 WT mice or AM14 3d mice were stimulated as described in part B but c-rel translocation to the nucleus was analyzed by confocal microscopy. D, I{kappa}B-{alpha} degradation. AM14 B cells were preincubated for 1 h with 50 µg/ml cycloheximide and stimulated with preformed chromatin ICs in the presence or absence of chloroquine. Cell lysates were electrophoresed and immunoblots were analyzed by densitometry. Levels of I{kappa}B-{alpha} were normalized by content of {alpha}-tubulin. Values are mean ± SEM of three different experiments.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The current report examined the functional outcome of chromatin IC activation of AM14 B cells and identified unique patterns of gene expression that included unanticipated effects on cytokine production. For example, both chromatin ICs and the combination of anti-IgM F(ab')2 plus CpG induced IL-2 expression. Although IL-2 is commonly considered a T cell cytokine, production of IL-2 has been noted previously in B cells activated by a combination of the BCR and CD40 (21) or by activated Th2 cells and Ag (22). The requirement for dual receptor engagement is similar to the situation in T cells where engagement of both the TCR and costimulatory receptors are necessary for optimal IL-2 production (13). In T cells, members of at least four unrelated families of transcription factors, NFAT, Oct, NF-{kappa}B, and AP-1, have been implicated in IL-2 production (23), consistent with the need for convergence of multiple signal transduction pathways for maximal gene expression. IL-2 can serve as a growth factor for both B cells and T cells and therefore the selective production of IL-2 by BCR/TLR9 engagement may play an important role in the prolonged survival of autoreactive lymphocytes.

In contrast to the IL-2 response, IL-6, IL-10, and IL-12p40 were induced by CpG (or other TLR ligands) (13), either alone or in combination with anti-IgM F(ab')2, but not by PL2–3. The lack of production of these cytokines by PL2–3-stimulated cells may be the consequence of multiple factors, including active suppression through the BCR or suboptimal stimulation of TLR9 by PL2–3. The high level of T-bet expression in TLR9-sufficient (but not TLR9-deficient) PL2–3-stimulated cells indicated that at least the pathway leading to the expression of this transcription factor was comparably activated by CpG, and chromatin ICs. Nevertheless, it is possible that other pathways downstream of TLR9 may be differentially triggered by the two types of ligand.

In plasmacytoid dendritic cells, the nature of the TLR9 response depends on the intracellular compartment in which the ligand and TLR9 interact and the site of interaction depends on the size of the ligand (24, 25). Thus, CpG-A ODNs form multimers and localize predominantly in early endosomes where TLR9 engagement leads to IFN-{alpha} production while CpG-B ODNs do not multimerize and are more likely to engage TLR9 in a late endosome and trigger IL-6 production. It is possible that chromatin ICs are delivered to one type of endosomal compartment where receptor interaction with other proteins might preferentially lead to IL-2 production, while the CpG used in this study (a CpG-B type) preferentially localizes to a different compartment where it triggers IL-6 production. We had previously noted that PL2–3 activation of AM14 B cells was much more sensitive to low concentrations of bafilomycin A (an agent that blocks endosome acidification) than was CpG activation; whether the differential response to subtle changes in pH reflects the site of ligand/TLR9 interaction remains to be determined.

BCR signaling in response to anti-IgM F(ab')2 and other high-affinity ligands has been extensively investigated, but the signaling components activated in response to lower affinity ligands are incompletely understood. We separated the BCR-signaling events triggered by chromatin ICs from TLR9/7-dependent signaling events by using AM14 B cells that lacked a functional TLR9 (or TLR9/7) signaling cascade. These studies included AM14 mice with targeted mutations in TLR9, MyD88, or a spontaneous mutation in Unc9B (8). The quality of the signal induced by chromatin ICs was inherently different from that induced by cross-linking the receptor with anti-IgM F(ab')2, a conventional, but nonphysiological, ligand. Although the overall pattern of tyrosine-phosphorylated proteins was similar, stimulation with 5 µg/ml PL2–3, the optimal mitogenic concentration, routinely resulted in delayed kinetics of phosphorylation and a less robust response than seen with anti-IgM F(ab')2. More rapid and extensive levels of tyrosine phosphorylation could be obtained with a higher concentration (20 µg/ml) of PL2–3, but even this concentration fails to induce proliferation, as measured by [3H]thymidine incorporation, in MyD88-deficient B cells as well as in 3d B cells.

The level of protein phosphorylation is the result of a balance between the action of kinases and phosphatases. We cannot rule out that the possibility that the less extensive tyrosine phosphorylation and reduced calcium flux might be a consequence of higher phosphatase activity. B cells express multiple inhibitory receptors, including Fc{gamma}RIIB (26). Coligation of BCR and Fc{gamma}RIIB can lead to negative regulation of the BCR-signaling cascade through recruitment of the inositol phosphatase SHIP (27). Chromatin ICs can potentially engage Fc{gamma}RIIB receptor. However, IgG2a is a weak ligand for Fc{gamma}RIIB and we have found that AM14 and AM14Fc{gamma}RIIB-deficient B cells proliferate comparably in response to PL2–3 through a TLR9-dependent mechanism and did not show major differences in the pattern of tyrosine phosphorylation (A. Avalos and A. Marshak-Rothstein, manuscript in preparation).

BCR engagement drives the assembly of a multiprotein scaffold, referred to as an "early signalosome," that is sufficient to trigger limited Ca2+ mobilization and activate NFAT and ERK (28), similar to the outcome observed in AM14 B cells in response to PL2–3. The current report demonstrates that BCR-dependent ERK phosphorylation is essential for the subsequent BCR/TLR9-dependent cell cycle entry of chromatin IC-activated AM14 B cells. In this context, ERK phosphorylation does not actively contribute to the form of anergy observed with higher affinity BCR ligands (29).

Formation of the early signalosome is not sufficient to drive B cell proliferation. NF-{kappa}B activation requires further assembly of adaptor proteins to form an "immunosome." Immunosome assembly is purportedly driven by extensive BCR cross-linking or, when there is less extensive cross-linking of the BCR, by the engagement of a costimulatory receptor (28). In the case of AM14 B cells stimulated by chromatin IC, the costimulus is likely to be TLR9. How the BCR and TLR9 intersect to form the functional equivalent of an immunosome is unclear. Some receptors, such as the epidermal growth factor receptor, have been found to activate the ERK pathway after sequestration to endosomal compartments (30, 31). Although BCR signaling and internalization have been considered mutually exclusive events (32), in our system, ERK activation is greatly reduced in the presence of inhibitors that block receptor internalization (L. Busconi, unpublished data). It is tempting to speculate that BCR-mediated internalization of early signalosome/immunosome components could lead to TLR9 colocalization in an endosomal compartment.

PL2–3 activation of AM14 B cells was suboptimal as determined by induced changes in intracellular Ca2+. PLC{gamma}2 generates two messengers, diacylglycerol and inositol (1, 4, 5)-triphosphate (IP3). IP3 then induces Ca2+ release from intracellular stores. However, PLC{gamma}2 is not only responsible for the transient initial phase of Ca2+ released from internal stores of the endoplasmic reticulum but also plays a role in the sustained second phase of Ca2+ entry from the extracellular space which is important for the amplification of BCR signaling (33). Because PLC{gamma}2 catalyzes the step immediately upstream of the increase in intracellular calcium, the weak PLC{gamma}2 activation that we observed may account for the suboptimal level of calcium flux and the lack of a sustained response (34).

In summary, chromatin ICs engage both the BCR and TLR9, although the overall strength of the each event may not be optimal. Nevertheless, this type of BCR/TLR9 dual receptor engagement leads to unique functional outcomes. A similar level of BCR/TLR cross-talk is likely to occur in any autoreactive B cell with a receptor specific for DNA or DNA-associated proteins or B cells reactive with RNA or RNA-associated proteins. The critical question to be addressed in future studies will be whether genes specifically induced by BCR/TLR coengagement will serve as useful therapeutic targets for the treatment of autoimmune diseases.


   Acknowledgments
 
We thank Dr. L. Glimcher (Harvard School of Public Health, Boston, MA) for providing the polyclonal antiserum specific for T-bet, Dr. S. Akira (Research Institute for Microbial Diseases, Osaka University, Osaka, Japan) for providing the MyD88–/– and TLR9–/– mice, and Dr. B. Beutler (The Scripps Research Institute, La Jolla, CA) for providing the 3d mice.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants AR-050256 (to A.M.-R.), AI-229690 (to T.L.R.), AI-50844 (to F.E.L.), and grants from the Alliance for Lupus Research (to T.W.B. and A.M.-R.). Back

2 Address correspondence and reprint requests to Dr. Ann Marshak-Rothstein, Department of Microbiology, Boston University School of Medicine, 80 East Concord Street, Boston, MA 02118. E-mail address: amrothst{at}bu.edu Back

3 Abbreviations used in this paper: IC, immune complex; ODN, oligodeoxynucleotide; PLC, phospholipase C; CsA, cyclosporin A; TNP, trinitrophenol. Back

Received for publication April 30, 2007. Accepted for publication September 6, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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