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B Cell Receptor Cross-Talk: Exposure to Lipopolysaccharide Induces an Alternate Pathway for B Cell Receptor-Induced ERK Phosphorylation and NF-B Activation¹

John R. Dye^*,, Arkadiy Palvanov^¶, Benchang Guo^,,¶ and Thomas L. Rothstein^2,,,,¶

Departments of ^* Pathology, Medicine, and Microbiology, Boston University School of Medicine, Boston, MA 02118; Immunobiology Unit, Evans Memorial Department of Clinical Research, Boston University Medical Center, Boston, MA 02118; and ^¶ Center for Oncology and Cell Biology, Feinstein Institute for Medical Research, Manhasset, NY 11030

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
BCR signaling in naive B cells depends on the function of signalosome mediators; however, prior engagement of CD40 or of IL-4R produces an alternate signaling pathway in which Bruton’s tyrosine kinase, PI3K, phospholipase C2, and protein kinase C are no longer required for BCR-induced downstream events. To explore the range of mediators capable of producing such an alternate pathway for BCR signaling, we examined the TLR4 agonist, LPS. B cell treatment with LPS at relatively low doses altered subsequent BCR signaling such that ERK phosphorylation and NF-B activation occurred in a PI3K-independent manner. This effect of LPS extended to MEK phosphorylation and IB degradation, and it developed slowly over a period of 16–24 h. The involvement of TLRs is suggested by similar effects observed with a structurally distinct TLR agonist, PAM3CSK4 and by the need for MyD88 for induction of alternate BCR signaling by LPS. Thus, LPS-mediated TLR engagement produces an alternate pathway for BCR-triggered signal propagation that differs from the classical, signalosome-dependent pathway.

	Introduction

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Signalosome elements are required for BCR signaling via the classical pathway (1); thus, interference with, or ablation of, any of PI3K, Bruton’s tyrosine kinase, B cell linker protein, phospholipase C (PLC)2,³ or protein kinase C (PKC) leads to a block in BCR-triggered downstream events. However, recent evidence indicates that following exposure to CD40L or IL-4, alternate pathways for BCR signaling are established in which downstream events take place without the need for PI3K and other signalosome members through a process of receptor cross-talk (2, 3, 4, 5). Thus, the generally accepted need for signalosome elements in BCR signaling may simply represent an initial condition applicable only to naive cells, and not to B cells in the midst of an immune response. The induction of alternate BCR signaling by T cell products raises the possibility that other factors associated with immune system activation or danger might do the same.

Along with other cellular constituents of the immune system, B cells express pattern recognition receptors in the form of TLRs (6). These receptors recognize a variety of foreign components, including Gram-positive and Gram-negative bacterial products, bacterial flagellin, and bacterially derived unmethylated CpG motifs (6). In particular, Gram-negative bacterial LPS is recognized by TLR4 (7). Substantial morbidity and mortality result from Gram-negative bacterial infections and sepsis (8). To more fully understand the immune response to Gram-negative bacterial invasion, we questioned whether bacterial LPS is capable of altering BCR signaling so that it proceeds via a less stringent, signalosome-independent alternate pathway.

The BCR signaling outcomes of ERK phosphorylation, a key step in AP-1 generation, and NF-B activation are differentially reliant on Bam32 and Carma1 and thus appear to represent distinct BCR signaling outcomes, although both are PI3K- and PKC-dependent (9, 10, 11, 12, 13, 14, 15). To determine whether LPS induces an alternate intracellular signaling pathway, we examined BCR-triggered phosphorylation of ERK and activation of NF-B in the presence of signalosome inhibitors.

	Materials and Methods

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Animals

Male BALB/cByJ mice and C57BL/6 mice at 6–8 wk of age were obtained from The Jackson Laboratory. MyD88^–/– and MyD88^+/– mice were kindly provided by Dr. Ann Marshak-Rothstein (Boston University School of Medicine, Boston, MA). Mice were cared for and handled in accordance with National Institute of Health and institutional guidelines.

B cell isolation

B cells from BALB/cByJ mice were prepared from spleen cell suspensions by negative selection as previously described (16). Briefly, splenocytes were depleted of T cells by treatment with anti-Thy-1.2 Ab, followed by complement lysis; the resultant cells were then subjected to density separation using Lympholyte M (Cedarlane Laboratories) to remove dead cells and RBC. B cells from MyD88^–/– and control mice were purified by negative selection using biotinylated Abs and streptavidin magnetic beads (Miltenyi Biotec). B cells were cultured at 2 x 10⁶/ml in RPMI 1640 (BioWhittaker) supplemented with 5% heat-inactivated FBS (Sigma-Aldrich), 10 mM HEPES (pH 7.25), 50 µM 2-ME, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin.

B cell stimulation

B cells were incubated in medium for 2 h (naive) and then stimulated by F(ab')₂ goat anti-mouse IgM (anti-Ig) or were treated with LPS (or other TLR agonist) for 24 h, washed, incubated in medium for 3 h, and then stimulated by anti-Ig. Inhibitors were added 1 h before stimulation with anti-Ig.

Western immunoblot analysis

Proteins were extracted from B cell pellets with RIPA lysis buffer. Nuclear and cytoplasmic fractions were separately extracted from B cells with the nuclear extraction kit obtained from Active Motif according to the manufacturer’s instructions. In each experiment, equal amounts of protein for each condition (15–30 µg) were subjected to SDS-PAGE followed by immunoblotting as previously described (17). Immunoreactive proteins were detected by ECL (Amersham Biosciences). Immunoblots were stripped and reprobed with control Ab to verify that equal amounts of protein were loaded in each lane.

Fractionation of LPS-stimulated B cells

B cells stimulated with LPS for 24 h were stained with fluorescence-labeled Abs to B220 and CD138, after which CD138^– B cells were obtained by FACS using a FACSAria sorter (BD Biosciences). Data were analyzed using FloJo software (Treestar).

Reagents

LPS from Salmonella typhimurium was obtained from Sigma-Aldrich. Affinity-purified F(ab')₂ polyclonal anti-Ig was obtained from Jackson ImmunoResearch Laboratories. Anti-phospho-Akt (Ser⁴⁷³), anti-Akt, anti-phospho-ERK1/2 (Thr²⁰²/Tyr²⁰⁴), anti-ERK1/2, anti-phospho-MEK1/2 (Ser²¹⁷/Ser²²¹), anti-MEK, and secondary Abs for immunoblotting were obtained from Cell Signaling Technology. Anti-IB and anti-c-Rel Abs were obtained from Santa Cruz Biotechnology. Anti-actin Ab was obtained from Sigma-Aldrich. LY294002, wortmannin, U73122, PP2, PP3, and rottlerin were obtained from Calbiochem. U0126 was obtained from Promega. Cycloheximide was obtained from Sigma-Aldrich. PAM3CSK4 was obtained from Invivogen. Recombinant murine IL-4, FITC-labeled anti-B220, and PE-labeled anti-CD138 were obtained from BD Pharmingen.

	Results

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BCR-induced ERK phosphorylation becomes resistant to LY294002 after B cell treatment with LPS

We investigated the possibility that bacterial products such as LPS might create an alternate BCR signaling pathway for MAPK activation similar to the alternate BCR signaling pathways established by B cell exposure to IL-4 and CD40L (2, 3, 4, 5). To evaluate this possibility, purified primary splenic B cells from normal BALB/c mice were treated with LPS for 24 h, washed, incubated in medium for 3 h ("rested"), and stimulated then with anti-Ig (15 µg/ml) after which whole cell extracts were prepared and examined by Western blotting using ERK-specific and phospho-ERK (pERK)-specific Abs. LPS-treated B cells were compared with purified primary B cells incubated in medium for 2 h (naive) and then stimulated with anti-Ig and Western blotted. We determined the PI3K dependence of BCR-induced ERK phosphorylation in each case by treating B cells with the specific inhibitor, LY294002 (20 µM), or control DMSO (the diluent for LY294002), for 1 h before addition of anti-Ig (Fig. 1).

View larger version (37K): [in this window] [in a new window]   FIGURE 1. BCR-induced ERK phosphorylation becomes resistant to LY294002 after B cell treatment with LPS. A, B cells were cultured in medium alone for 2 h (MED) or with LPS at 25 µg/ml for 24 h after which B cells were stimulated with F(ab')2 fragments of goat anti-mouse IgM Ab at 15 µg/ml (anti-Ig) for the indicated times. B cells were exposed to the PI3K inhibitor, LY294002 at 20 µM, or diluent control DMSO starting 60 min before addition of anti-Ig. Whole cell extracts were prepared and Western blotted as described in Materials and Methods with anti-phospho-ERK Ab. Blots were stripped and reprobed with anti-ERK Ab to control for variation in loading of extracted protein. One of four comparable experiments is shown. B, B cells were treated as described above using LPS at doses between 0.01 and 1 µg/ml, as indicated, and then stimulated with anti-Ig for 5 min. Whole cell extracts were Western blotted with anti-phospho-ERK Ab, after which blots were stripped and reprobed with anti-ERK Ab. One of three experiments with comparable results is shown. C, B cells were treated as described before using LPS at 1 µg/ml and LY294002 at several doses, as indicated (0 = B cells treated with diluent control DMSO). B cells were stimulated with anti-Ig for 5 min. Whole cell extracts were prepared and Western blotted with anti-phospho-Akt Ab, after which blots were stripped and reprobed with anti-Akt Ab. One of two comparable experiments is shown.

Initial experiments were conducted with a fully stimulatory dose of LPS at 25 µg/ml, as noted above. To determine the minimum amount of LPS capable of altering subsequent BCR signaling, we treated purified B cells with decreasing doses of LPS before stimulation with anti-Ig, which was in turn conducted in the presence or absence of PI3K inhibition. B cell treatment with LPS at 1 µg/ml yielded BCR-induced ERK phosphorylation that was LY294002 resistant, much like treatment with LPS at 25 µg/ml. However, induction of LY294002 resistance in BCR signaling waned when B cells were pretreated with a lower dose of LPS at 0.01 µg/ml (Fig. 1B). All subsequent experiments were conducted with LPS at 1 µg/ml.

To rule out the possibility that LY294002 failed to penetrate, or was inactive in, LPS-treated B cells, we examined the PI3K substrate, Akt (18). B cells were treated with LPS for 24 h, washed, incubated in medium for 3 h ("rested"), and stimulated then with anti-Ig (15 µg/ml) after which whole cell extracts were prepared and examined by Western blotting using Akt-specific and phospho-Akt (pAkt)-specific Abs. LPS-treated B cells were compared with B cells incubated in medium for 2 h (naive) and then stimulated with anti-Ig and Western blotted. B cells were treated with DMSO or with LY294002 before stimulation with anti-Ig. BCR triggering produced substantial Akt phosphorylation in both naive and LPS-treated B cells, and increasing doses of LY294002 inhibited anti-Ig-induced pAkt in LPS-treated B cells similarly to medium-cultured B cells (Fig. 1C). Importantly, the dose of LY294002 used in the experiments presented in this report, 20 µM, completely blocked PI3K-induced Akt phosphorylation in LPS-treated B cells. Thus, LPS treatment produces an alternate pathway for BCR signaling that operates independently of PI3K. This conclusion is supported by experiments in which a structurally unrelated PI3K inhibitor, wortmannin, blocked anti-Ig-induced pERK in naive but not in LPS-treated B cells (data not shown). Similar results obtained with U73122, a specific inhibitor of PLC, suggest that LPS-induced reprogramming allows BCR signaling to bypass the need for additional signalosome elements (data not shown).

LPS-mediated reprogramming of BCR signaling for ERK phosphorylation is time dependent

To determine the speed with which LPS affects subsequent BCR signaling, we treated B cells with LPS at 1 µg/ml for various periods of time before stimulation with anti-Ig, in the presence or absence of LY294002 (Fig. 2). B cell treatment with LPS for 24 h produced LY294002 resistance in subsequent BCR signaling for ERK phosphorylation, whereas treatment for 4 h failed to do so. B cell treatment with LPS for longer periods of 48–72 h was not different than treatment for 24 h (data not shown). B cell treatment for 16 h produced minimal PI3K-independent BCR-triggered pERK. Thus, B cell treatment with LPS for 16–24 h is required for generation of a PI3K-independent BCR signaling pathway. In parallel experiments, LPS treatment for 16–24 h, but not for 4 h, produced resistance to PLC inhibition with U73122 in subsequent BCR signaling for ERK phosphorylation (data not shown).

View larger version (48K): [in this window] [in a new window]   FIGURE 2. LPS-mediated reprogramming of BCR signaling for ERK phosphorylation is time dependent. B cells were cultured in medium alone for 2 h (MED) or with LPS at 1 µg/ml for various periods of time, as indicated, after which B cells were stimulated with anti-Ig at 15 µg/ml for 5 min. B cells were exposed to the PI3K inhibitor, LY294002 at 20 µM (LY), or diluent control DMSO (DM) starting 60 min before addition of anti-Ig. Whole cell extracts were prepared and Western blotted with anti-phospho-ERK Ab, after which blots were stripped and reprobed with anti-ERK Ab to control for variation in loading of extracted protein. One of three comparable experiments is shown.

To confirm that LPS induction of an alternate pathway for BCR signaling depends on TLR triggering, we examined MyD88-deficient B cells. We stimulated untreated and LPS-treated (1 µg/ml) MyD88-deficient and control C57BL/6 B cells with anti-Ig, in the presence or absence of LY294002 and then evaluated lysates for the presence of pERK (Fig. 3). Control B cells behaved as described above. LPS treatment produced resistance to LY294002-mediated inhibition of subsequent BCR-triggered ERK phosphorylation which was apparent in untreated B cells. However, the situation was quite different for MyD88-deficent B cells; here, LPS treatment failed to alter LY294002 inhibition of BCR-triggered pERK. Thus, induction of PI3K independence in BCR signaling by LPS requires MyD88 and thus likely depends on the TLR/MyD88 signaling pathway.

View larger version (36K): [in this window] [in a new window]   FIGURE 3. LPS-induced reprogramming of BCR signaling fails in MyD88-deficient B cells. B cells were obtained from MyD88-deficient (MyD88 KO) and normal C57BL6 (Control) mice and were cultured in medium alone for 2 h (MED) or with LPS at 1 µg/ml for 24 h after which B cells were stimulated with anti-Ig at 15 µg/ml for 15 min. B cells were exposed to the PI3K inhibitor, LY294002 at 20 µM, or diluent control DMSO starting 60 min before addition of anti-Ig. Whole cell extracts were prepared and Western blotted with anti-phospho-ERK Ab. Blots were stripped and reprobed with anti-ERK Ab. One of two comparable experiments is shown.

In the classical BCR signaling pathway, ERK phosphorylation is directly dependent on activation of the upstream MAPK kinase, MEK (19). To determine whether BCR signaling to pERK involves MEK in the alternate pathway, we stimulated untreated and LPS-treated B cells with anti-Ig, in the presence or absence of LY294002, and then evaluated lysates for the presence of pMEK (Fig. 3A). Anti-Ig stimulation of control (medium) B cells produced MEK phosphorylation within 5 min, in keeping with previous work (20). This BCR-induced MEK phosphorylation was highly sensitive to inhibition of PI3K and was largely blocked by B cell exposure to LY294002. However, LPS markedly altered the MEK response to BCR engagement. After LPS treatment, a large proportion of anti-Ig-induced MEK phosphorylation became resistant to inhibition by LY294002. Thus, LPS treatment alters the signaling requirements for BCR-induced MEK phosphorylation just as it does for BCR-induced ERK phosphorylation (Fig. 4).

View larger version (52K): [in this window] [in a new window]   FIGURE 4. BCR-induced MEK phosphorylation becomes substantially PI3K independent after B cell treatment with LPS, and MEK is required for LPS-induced reprogramming of BCR signaling for ERK phosphorylation. A, B cells were treated as described in the legend to Fig. 1A. Whole cell extracts were prepared and Western blotted with anti-phospho-MEK Ab. Blots were stripped and reprobed with anti-MEK Ab. One of three comparable experiments is shown. B, B cells were cultured in medium alone for 2 h (MED) or with LPS at 1 µg/ml for 24 h, after which B cells were stimulated with anti-Ig at 15 µg/ml for the indicated times. B cells were exposed to the MEK inhibitor, U0126 at 10 µM, or diluent control DMSO starting 60 min before addition of anti-Ig. Whole cell extracts were prepared and Western blotted with anti-phospho-ERK Ab. Blots were stripped and reprobed with anti-ERK Ab. One of two comparable experiments is shown.

The LPS-induced alternate pathway for BCR signaling depends on src kinase activity but is not inhibited by rottlerin

We further evaluated the requirements for the LPS-induced alternate pathway by determining reliance on src kinase activity. We blocked src kinases with the inhibitor PP2 and compared BCR-triggered ERK phosphorylation with results obtained using the inactive analog, PP3. These reagents were added prior to anti-Ig stimulation of untreated control B cells and LY294002-inhibited, LPS-treated B cells, in which the only BCR signaling pathway operative was the alternate pathway. In control B cells, src kinase inhibition with PP2 eliminated BCR signaling for pERK, whereas PP3 had no effect, as expected. The same was true for LPS-treated B cells in which PI3K was blocked by LY294002, indicating that the LPS-induced alternate pathway for BCR signaling, like the classical pathway, depends on src kinase activity (Fig. 5).

View larger version (41K): [in this window] [in a new window]   FIGURE 5. The LPS-induced alternate pathway for BCR signaling depends on src kinase activity but is not inhibited by rottlerin. B cells were cultured in medium alone for 2 h (MED), or with LPS at 1 µg/ml for 24 h, or with IL-4 at 10 ng/ml, after which B cells were stimulated with anti-Ig at 15 µg/ml for 15 min. B cells were exposed to LY294002 at 20 µM alone or in combination with PP2 at 20 µM, PP3 at 20 µM, or rottlerin at 10 µM, as indicated, starting 60 min before addition of anti-Ig. Whole cell extracts were prepared and Western blotted with anti-phospho-ERK Ab. Blots were stripped and reprobed with anti-ERK Ab. One of three comparable experiments is shown.

The LPS-induced alternate pathway for BCR signaling requires a higher dose of anti-Ig than the classical pathway

To determine the strength of signal required to produce ERK phosphorylation via the alternate and classical pathways, we titrated the dose of anti-Ig added to LPS-stimulated B cells in the presence or absence of LY294002. As noted above, when B cells were stimulated with anti-Ig at 15 µg/ml, substantial amounts of BCR-triggered pERK were resistant to inhibition with LY294002, indicating operation of the LPS-induced alternate, PI3K-independent signaling pathway. However, as the dose of anti-Ig was reduced to 0.6 µg/ml, resistance to LY294002 in BCR-triggered pERK was lost (Fig. 6). Thus, at lower doses of anti-Ig, the classical pathway leading to ERK phosphorylation was triggered, but the LPS-induced, LY294002-resistant, alternate pathway was not.

View larger version (30K): [in this window] [in a new window]   FIGURE 6. The LPS-induced alternate pathway for BCR signaling requires a higher dose of anti-Ig than the classical pathway. B cells were cultured in medium alone for 2 h (MED), or with LPS at 1 µg/ml for 24 h, after which B cells were stimulated with anti-Ig at the indicated doses for 15 min. B cells were exposed to LY294002 at 20 µM as indicated starting 60 min before addition of anti-Ig. Whole cell extracts were prepared and Western blotted with anti-phospho-ERK Ab. Blots were stripped and reprobed with anti-ERK Ab. One of two comparable experiments is shown.

LPS is well known to stimulate Ig secretion on the part of B cells, for which reason it might be thought that the alternate pathway for BCR signaling corresponds to a differentiated B cell stage. To address this issue, we treated B cells with LPS as above and then sorted nondifferentiated CD138^– B cells. In two experiments, 6–9% of LPS-treated B cells were CD138⁺ (presort), and isolated CD138^– B cells were 99.5% pure (postsort). B cells were then stimulated with anti-Ig in the presence or absence of LY294002. CD138^– LPS-treated B cells behaved as noted above for LPS-treated B cells; that is, anti-Ig-induced ERK phosphorylation was substantially resistant to inhibition by LY294002 (Fig. 7). Thus, B cells in which LPS treatment had not produced differentiation, as reflected in the lack of acquisition of CD138, manifested the alternate pathway similarly to unseparated, LPS-treated B cells. These results strongly suggest that reprogramming of the BCR signaling pathway produced by LPS is not correlated with B cell differentiation toward the plasma cell stage.

View larger version (51K): [in this window] [in a new window]   FIGURE 7. LPS-induced reprogramming of BCR signaling does not depend on differentiation. B cells were cultured in medium alone for 2 h (MED), or with LPS at 1 µg/ml for 24 h, after which B cells were sorted on the basis of CD138 expression (CD138-). B cells were stimulated with anti-Ig at 15 µg/ml for 15 min in the presence or absence of LY294002 at 20 µM, as indicated, starting 60 min before addition of anti-Ig. Whole cell extracts were prepared and Western blotted with anti-phospho-ERK Ab. Blots were stripped and reprobed with anti-ERK Ab. One of two comparable experiments is shown.

LPS reprograms BCR signaling for NF-B activation in addition to ERK phosphorylation

We questioned whether LPS treatment influences other BCR signaling pathways and in so doing we focused on BCR-triggered activation of NF-B, which has been shown previously to be affected by CD40L, but not by IL-4 (2, 4, 5). To evaluate NF-B activation, we determined the level of nuclear c-Rel under various conditions, inasmuch as BCR-induced nuclear NF-B consists primarily of p50 and c-Rel (24, 25, 26). B cells were treated with LPS for 24 h and then washed and cultured in medium for 24 additional hours to re-establish baseline levels of NF-B. Untreated control and LPS-treated B cells were stimulated with anti-Ig for 3 h in the presence or absence of LY294002 after which cells were lysed, nuclei collected, and Western blotting was conducted (Fig. 8A). In control B cells, anti-Ig stimulation produced an increase in nuclear c-Rel that was blocked by LY294002. In LPS-treated B cells, however, anti-Ig stimulation produced an increase in nuclear c-Rel that was not blocked by LY294002. Thus, following LPS treatment, BCR-triggered NF-B activation no longer exhibits sensitivity to LY294002 and thus has become PI3K independent.

View larger version (51K): [in this window] [in a new window]   FIGURE 8. LPS reprograms BCR signaling for NF-B activation in addition to ERK phosphorylation. A, B cells were cultured in medium alone for 2 h (MED) or with LPS at 1 µg/ml for 24 h, after which B cells were stimulated with anti-Ig at 15 µg/ml for 3 h. B cells were exposed to the PI3K inhibitor, LY294002 at 20 µM (LY), or diluent control DMSO (DM), starting 60 min before addition of anti-Ig. Cells were lysed, and nuclear extracts were Western blotted with anti-c-Rel Ab. Blots were stripped and reprobed with anti-B1-lamin Ab to control for variation in loading of extracted protein, and with anti--tubulin Ab to evaluate cytosolic contamination. A whole cell extract of unstimulated B cells (CE) was used as a positive control. One of three comparable experiments is shown. B, B cells were cultured in medium alone for 2 h (MED) or with LPS at 1 µg/ml for 24 h, after which B cells were stimulated with anti-Ig at 15 µg/ml for the times indicated. B cells were exposed to cycloheximide at 50 µM plus the PI3K inhibitor, LY294002 at 20 µM (LY), or diluent control DMSO (DM), starting 60 min before addition of anti-Ig. Whole cell extracts were prepared and Western blotted wth anti-IB Ab, after which blots were stripped and reprobed with anti-actin Ab. One of two comparable experiments is shown.

PAM3CSK4 reprograms BCR signaling for ERK phosphorylation and NF-B activation

To determine whether LPS induction of an alternate pathway for BCR signaling extends to other TLR agonists, we examined PAM3CSK4, a synthetic tripalmitoylated lipopeptide that mimics the acylated amino termini of bacterial lipoproteins and binds to TLR2/1 heterodimers (28). We stimulated untreated control and PAM3CSK4-treated (600 ng/ml) B cells with anti-Ig, in the presence or absence of LY294002, and then evaluated lysates for the presence of pERK (Fig. 9A). In contrast to control B cells, in PAM3CSK4-treated B cells a substantial portion of anti-Ig-induced ERK phosphorylation was resistant to inhibition by LY294002. Thus, like LPS treatment, PAM3CSK4 treatment alters the signaling requirements for BCR-induced ERK phosphorylation.

View larger version (45K): [in this window] [in a new window]   FIGURE 9. PAM3CSK4 reprograms BCR signaling for ERK phosphorylation and NF-B activation. A, B cells were cultured in medium alone for 2 h (MED) or with LPS at 1 µg/ml or with PAM3CSK4 at 600 ng/ml (PAM) for 24 h, after which B cells were stimulated with anti-Ig at 15 µg/ml for 5 min. B cells were exposed to the PI3K inhibitor, LY294002 at 20 µM (LY) or to diluent control DMSO (DM), starting 60 min before addition of anti-Ig. Whole cell extracts were prepared and Western blotted wth anti-phospho-ERK Ab, after which blots were stripped and reprobed with anti-ERK Ab. B, B cells were cultured in medium alone (MED) or with PAM3CSK4 at 600 ng/ml (PAM) as described above. B cells were exposed to cycloheximide at 50 µM plus the PI3K inhibitor, LY294002 at 20 µM (LY), or diluent control DMSO (DM), starting 60 min before addition of anti-Ig. Whole cell extracts were prepared and Western blotted with anti-IB Ab, after which blots were stripped and reprobed with anti-actin Ab. One of three comparable experiments is shown.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The present study demonstrates that LPS treatment establishes a new, alternate pathway for BCR-triggered ERK phosphorylation and NF-B activation that operates independently of PI3K, in direct contrast to the PI3K-dependence of BCR signaling in naive B cells, and thus LPS reprograms the connection between BCR and downstream events. This reprogramming requires TLR signaling through MyD88 and is mimicked by additional TLR agonists, including PAM3CSK4 and CpG DNA (unpublished observations). The alternate pathway relies on src kinase activity but essentially bypasses PI3K, and presumably other signalosome elements, ultimately intersecting the classical pathway at some point beyond the signalosome because in the alternate pathway, as in the classical pathway, MEK is responsible for ERK phosphorylation and IB degradation is associated with NF-B activation.

In work carried out to date, we have delineated two distinct alternate pathways for BCR signal propagation produced by receptor crosstalk. In the first, PI3K-independence extends to MEK/ERK and IB/NF-B. This pathway is induced by CD40L (2, 3, 4). In the second, PI3K-independence extends to MEK/ERK but not to NF-B. This pathway is induced by IL-4 (5). The LPS-induced (and PAM3CSK4-induced) alternate pathway for BCR signaling promotes activation of both ERK and NF-B, and thus appears to most closely parallel the CD40L-induced alternate pathway. Further evidence that the LPS-induced alternate pathway differs from the IL-4-induced alternate pathway lies is the sensitivity of the latter, but not the former, to inhibition by rottlerin. The differential reliance of MAPK and NF-B activation on Bam32 and Carma1 suggests a mechanism whereby alternate pathway ERK phosphorylation may be dissociated from, or combined with, activation of NF-B, depending on recruitment of these two mediators (9, 10, 11, 12, 13, 14, 15). Still the mediators that bridge BCR and MEK/IB without the need for PI3K remain obscure, although rottlerin resistance suggests that PKC is not involved, whereas the time required for induction of alternate BCR signaling by LPS suggests that new protein synthesis is involved. Unfortunately, the latter could not be tested directly because inhibitors of protein synthesis are toxic to primary B cells over the period of time required for induction of the LPS-induced alternate pathway.

Although no inhibitor is perfectly selective, the clear-cut difference in the LY294002 resistance of ERK and NF-B activation in LPS-treated vs untreated B cells speaks to LPS-induced alteration of BCR signaling. Moreover, the use of PI3K inhibition presents distinct advantages over the use of knockout animals, because B cells in the latter develop in an abnormal environment and as a result of endogenous or exogenous deficiencies may manifest compensatory changes to yield results that cannot be extrapolated to the normal situation, as recently reported (29, 30). Further, knockdown of PI3K with small interfering RNA is impractical in these kinds of experiments because of the potentially confounding effects of IFN production (31).

The LPS-induced alternate pathway is not a function of B cell differentiation. At the same time, triggering of the alternate pathway requires a higher degree of BCR cross-linking than does triggering of the classical pathway. These considerations suggest that following exposure to bacterial products in the form of TLR agonists, B cell responses can be augmented in the face of high doses of Ag. This may represent a backup strategy to enhance serological responses to Ag overload during bacterial infections.

That raises the question of whether naturally occurring LPS levels are sufficient to trigger the alternate pathway. Plasma LPS levels can rise above 10 ng/ml in patients with bacterial sepsis, although levels this high are associated with very high mortality (32). In the current in vitro experiments, LPS at 100 ng/ml produced PI3K-independent BCR signaling, although LPS at 10 ng/ml either failed to do so or, in some experiments, did so very weakly. Thus, the in vitro effect of LPS on B cell signaling occurs at a level that appears to lie just beyond the level of LPS observed in very ill patients. However, stimulation by LPS is highly dependent on CD14, and it is difficult to draw a parallel between the levels of CD14 present in vitro and in vivo (33, 34). Further, local levels of LPS and other bacterial products may be higher than systemic levels. With these caveats, our results suggest that receptor cross-talk is likely capable of influencing BCR signaling at levels of LPS that might occur during severe Gram-negative bacterial infections. Equally important, however, is the suggestion that this new, alternate pathway for BCR signaling induced by LPS and other TLR agonists may be responsible, at least in part, for enhancement of serological responses induced by immunization with CpG and/or other similar adjuvants.

	Acknowledgment

 
The authors thank Dr. Ann Marshak-Rothstein for providing MyD88- deficient 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 U.S. Public Health Service Grant AI40181 awarded by the National Institutes of Health. J.R.D. is the recipient of a Karen Grunebaum Fellowship in Cancer Research.

² Address correspondence and reprint requests to Dr. Thomas L. Rothstein, Center for Oncology and Cell Biology, The Feinstein Institute for Medical Research, 350 Community Drive, Manhasset, NY 11030. E-mail address: tr{at}nshs.edu

³ Abbreviations used in this paper: Akt, cellular homology of the AKR mouse T cell lymphoma-transforming retrovirus; PKC, protein kinase C; PLC, phospholipase C; PAM3CSK4, N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)propyl]-[R]-cysteinyl-[S]-seryl- [S]-lysyl-[S]-lysyl-[S]-lysyl-[S]-lysine · 3HCl; anti-Ig, F(ab')₂ goat anti-mouse IgM; pERK, phospho-ERK.

Received for publication August 18, 2006. Accepted for publication April 29, 2007.

	References

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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