BCR signaling is propagated by a series of intermediaries and eventuates in NF-κB activation, among other outcomes. Interruption of several mediators that constitute the signalosome, such as PI3K and phospholipase Cγ2, completely blocks BCR signaling for NF-κB. We show here that this accepted, conventional paradigm is, in fact, limited to naive B cells. CD40L treatment reprograms normal B cells such that a novel, alternate pathway for BCR signaling is created. Through this alternate pathway BCR triggering induces nuclear NF-κB without the need for PI3K or for phospholipase Cγ2. Induction of NF-κB via the alternate pathway is accompanied by IκB kinase β (IKKβ) phosphorylation, IκBα phosphorylation, and IκBα degradation, and inhibition of IKKβ blocked IκBα degradation. Several key events in the conventional pathway, including early protein tyrosine phosphorylation, were unimpeded by generation of the alternate pathway which appears to operate in parallel, rather than in competition, with classical BCR signaling. These results demonstrate cross-talk between CD40 and BCR, such that the requirements for BCR signaling are altered by prior B cell exposure to CD40L. The alternate BCR signaling pathway bypasses multiple signalosome elements and terminates in IKKβ activation.

Bcell receptor engagement is vital for the development, survival, differentiation, and immune responsiveness of B lymphocytes (1, 2, 3). Early steps in signal transduction triggered by BCR engagement depend on tyrosine kinase activity facilitated by protein-protein interactions specified by src homology 2 (SH2) 4 and SH3 domains, protein-lipid interactions mediated by pleckstrin homology (PH) domains, and membrane localization enhanced by posttranslational modifications. Principal tyrosine kinases include Lyn and possibly other src kinases, Syk, and Bruton’s tyrosine kinase (Btk). Syk is responsible for phosphorylating and activating several downstream mediators, including PI3K, B cell linker (BLNK), and, in conjunction with Btk, phospholipase C (PLC). PI3K facilitates activation of PH-containing mediators, such as Btk. PLC produces second messenger molecules that increase intracellular Ca2+ and activate protein kinase C (PKC).

Several of these molecules have been linked in a conceptual framework termed the signalosome (4). The prototype molecule for this group is Btk, mutation of which results in severely blocked B cell development in humans afflicted with X-linked agammaglobulinemia, but a milder form of B cell deficiency in mice with X-linked immunodeficiency. This deficiency includes reduced numbers of conventional B cells, virtually complete absence of B-1 cells, and low serum Ig levels (5, 6). Similar deficiencies have been described in mice constructed with genetic deficiencies of the p85α subunit of PI3K, the p110δ subunit of PI3K, BLNK, PLCγ2, and PKCβ (7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19). Thus, each of these molecules is essential for normal B cell development and survival. Some aspects of the B cell deficiencies that characterize these mice are likely due to reduced expression of survival genes resulting from impairment of BCR-induced NF-κB activation, which is found in each of these models (3).

NF-κB consists of hetero- and homodimers drawn from a panel of five mammalian proteins that each share the ∼300-aa Rel homology domain: RelA (p65), RelB, c-Rel, p50, and p52 (reviewed in Ref. 20). Although constitutively active in mature, primary B cells, NF-κB is further induced following BCR engagement, resulting in induction of κB-dependent gene expression (21, 22). NF-κB activation is regulated by a family of ankyrin repeat-containing inhibitor proteins that normally retain NF-κB dimers within the cytosol in an inactive state (reviewed in Ref. 23), the prototype for which, and most active member, is IκBα. Dissociation of NF-κB results from posttranslational modification and destruction of IκBα that begins with IκBα phosphorylation on serines 32 and 36, which targets IκBα for recognition by β-TrCP and subsequent ubiquitination, which targets IκBα for degradation in the proteasome.

The kinase responsible for phosphorylating IκBα is a large multicomponent complex of which two of the subunits, IκB kinase α (IKKα) and IKKβ, are related, catalytically active molecules, whereas a third component, IKKγ, is regulatory in function and unrelated to the other two in sequence (reviewed in Ref. 24). IKKα and IKKβ are phosphorylated on serines 176/180 and 177/181, respectively, although the responsible kinase has not been identified and the means by which signalosome elements are connected to IKK has not been clarified. IKKα and IKKβ display similar substrate specificities, although IKKβ appears to be a more potent IκBα kinase than IKKα and gene-targeting experiments indicate that IKKβ, and not IKKα, is necessary for NF-κB induction by proinflammatory stimuli (24, 25).

Our recent studies of the requirements for inducible resistance against Fas-mediated apoptosis in B cells led to elucidation of an alternate pathway for BCR signaling (26, 27, 28). We found that treatment of Btk mutant, xid B cells with CD40L markedly changed the BCR signaling requirements for IκBα degradation and NF-κB activation (28). In particular, whereas BCR-induced NF-κB activation failed in naive xid B cells (29), treatment with CD40L restored BCR-triggered NF-κB activation (28). These results showed the capacity of CD40L treatment to ameliorate the defect in BCR signaling produced by mutant Btk in xid B cells. However, B cells developing in the absence of a key signaling intermediary like Btk experience altered expression of other molecules (30), and those other molecules might constitute or contribute to an alternate pathway for BCR signaling that is found only in mutant B cells. Thus, initial results on xid B cells showing cross-talk-induced bypass of mutant Btk do not address the key question of whether reprogramming for an alternate pathway exists in normal B cells. Further, these initial results raise additional questions of whether other signalosome mediators may be similarly bypassed, and at what point this new pathway converges with the conventional pathway for activation of NF-κB.

PI3K phosphorylates phosphatidylinositol-(4,5)-bisphosphate (PIP2) to phosphatidylinositol-(3,4,5)-triphosphate (PIP3), which acts to recruit Btk to the membrane in a PH domain-dependent manner (along with other PH-containing molecules), resulting in the activation of Btk which in turn facilitates activation of PLCγ2 (31). PI3K also activates Akt in BCR signaling (32). These multiple functions suggest that PI3K has more extensive effects than Btk. The availability of specific PI3K inhibitors, such as LY294002 and wortmannin, provides the means to block PI3K activity in real time, and studies with these agents have demonstrated the PI3K-dependence of BCR-induced NF-κB activation (33). In view of the extensive role of PI3K in BCR-triggered signaling outcomes, we used these inhibitors to determine whether the CD40-induced alternate pathway for BCR signaling is idiosyncratic to xid B cells or exists in normal B cells. We now report that the alternate pathway is indeed produced by CD40 cross-talk in normal B cells, that it is capable of bypassing the need for PI3K and for PLCγ2 in BCR signaling, and that it produces phosphorylation of IKKβ on the way to triggering IκBα degradation and NF-κB activation.

Male BALB/cByJ mice at 6–8 wk of age were obtained from The Jackson Laboratory. Mice were cared for and handled in accordance with National Institutes of Health and institutional guidelines.

B cells were prepared form spleen cell suspensions by negative selection as previously described (34). 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 were cultured at 2–4 × 106/ml in RPMI 1640 medium (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 cells were stimulated by F(ab′)2 goat anti-mouse IgM (anti-Ig) with or without prior CD40L treatment. B cells stimulated without CD40L treatment (CD40L) were cultured in medium for 3 h before addition of anti-Ig. B cells stimulated with CD40L treatment (CD40L(+)) were cultured with soluble recombinant CD40L (see below) for 48 h (except as noted), washed with medium three times, then cultured in medium for 1 h before addition of anti-Ig. Inhibitors were added 30 min before stimulation with anti-Ig, except for jesterone dimer (JD), which was added 2 h before stimulation with anti-Ig.

Nuclear extracts were prepared and analyzed for binding to a radiolabeled κB site-containing oligonucleotide probe as previously described (28). Supershift analysis was conducted as previously described using Rel-specific Abs generously provided by Dr. N. Rice (National Cancer Institute, Frederick, MD) (28). Affinity-purified rabbit polyclonal STAT6-specific Ab (Santa Cruz Biotechnology) was used as a specificity control for supershift analysis.

Protein was extracted from cell pellets with 1% Nonidet P-40 lysis buffer containing protease inhibitors, and equal amounts (10–30 μg) were subjected to SDS-PAGE followed by immunoblotting as previously described (28). Anti-phospho-IKKα/IKKβ (Ser180/Ser181), anti-phospho-IκBα (Ser32/Ser36), anti-phospho-Akt (Ser473), and anti-Akt Abs were obtained from Cell Signaling Technology. Anti-IKKα, anti-IKKβ, anti-IκBα, anti-BLNK, and anti-PLCγ2 Abs were obtained from Santa Cruz Biotechnology. Anti-phosphotyrosine Ab (4G10) was obtained from Upstate Biotechnology. Rabbit or mouse secondary Abs were obtained from Jackson ImmunoResearch Laboratories. Immunoreactive proteins were detected by ECL (Amersham Biosciences). Blots were stripped and reprobed with anti-actin Ab (Sigma-Aldrich) to verify that equal amounts of protein were loaded in each lane.

Whole cell lysates were precleared with protein G Sepharose (Amersham Biosciences) and incubated with Abs as indicated for 2 h at 4°C, followed by further incubation with protein G-Sepharose and a lysis buffer wash. Resultant immunoprecipitates were subjected to SDS-PAGE and Western blotting as described above.

Affinity-purified F(ab′)2 of polyclonal goat anti-mouse IgM Ab (anti-Ig) was obtained from Jackson ImmunoResearch Laboratories. Preparation of soluble CD40L/CD8α (35) and cross-linking anti-CD8α Ab have been described (28, 36); these reagents were used at 1/10 and 1/40 dilutions of dialyzed supernatants, as described (36). PMA, ionomycin, and cycloheximide (CHX) were obtained from Sigma-Aldrich. LY294002, wortmannin, and U73122 were obtained from Calbiochem. Lactacystin was obtained from BIOMOL. JD (37) was kindly provided by Dr. J. A. Porco (Department of Chemistry, Boston University, Boston, MA).

We evaluated the possibility that CD40 engagement produces an alternate pathway for BCR signaling in normal B cells by making use of specific inhibitors to interfere with PI3K in real time. We stimulated purified B cells from normal BALB/c mice with anti-Ig in the presence or absence of the PI3K inhibitor, LY294002, with or without prior CD40L treatment. Nuclear extracts were prepared and analyzed for expression of NF-κB by EMSA. Results are shown in Fig. 1 A. Naive B cells constitutively expressed nuclear NF-κB, as expected. Treatment with anti-Ig produced a substantial increase in nuclear NF-κB in control (DMSO-treated) B cells, whereas anti-Ig completely failed to up-regulate NF-κB in B cells treated with LY294002, as expected (33, 38). B cells treated with LY294002 were not generally impaired in their NF-κB responses, because nuclear NF-κB was stimulated by the mitogenic combination of PMA and ionomycin, which does not depend on PI3K (data not shown). Thus, BCR engagement of naive B cells up-regulated nuclear NF-κB in a PI3K-dependent fashion, in accordance with previous reports using PI3K inhibitors (33) or PI3K knockout mice (38, 39).

FIGURE 1.

BCR engagement activates NF-κB in PI3K-inhibited B cells after treatment with CD40L. A, Anti-Ig induces nuclear κB-binding activity in CD40L-treated B cells in the presence of LY294002. B cells were cultured in medium (M) alone (CD40L(−)), or were stimulated with F(ab′)2 of goat anti-mouse IgM at 15 μg/ml (anti-Ig (αIg)) or with the combination of PMA and ionomycin at 100 and 600 ng/ml, respectively (P/I), for 3 h; B cells were cultured with CD40L/CD8α plus cross-linking anti-CD8 Ab for 48 h (CD40L+/M), or were cultured with CD40L and stimulated with αIg or P/I for the last 3 h of 48 h cultures. B cells were exposed to the PI3K inhibitor, LY294002 at 10 μM (LY), or diluent control DMSO (DM), starting 30 min before addition of anti-Ig or P/I. Nuclei were isolated and extracted as described in Materials and Methods. Nuclear extract protein was tested for binding to a κB site-containing oligonucleotide by EMSA. Binding to an NF-Y site-containing oligonucleotide was used to verify that equal amounts of nuclear extract protein were evaluated. The levels of nuclear κB-binding activity in anti-Ig-treated samples over medium control values after normalization to NF-Y are shown below the corresponding lanes. Results are shown for one of three comparable experiments. B, The composition of NF-κB induced by anti-Ig in CD40L-treated B cells is the same in the presence or absence of PI3K inhibition. Nuclear extract protein samples were obtained from unstimulated (M) or anti-Ig-stimulated (αIg) CD40L-treated B cells in the presence of LY294002 or of control DMSO, and subjected to EMSA, as described above. The identity of anti-Ig-induced κB-binding proteins was evaluated by including Abs that recognize p50, p52, p65, RelB, and c-Rel, as indicated, in the binding reactions to supershift nucleoprotein complexes. Anti-STAT6 Ab was used as a specificity control. One of two comparable experiments is shown.

FIGURE 1.

BCR engagement activates NF-κB in PI3K-inhibited B cells after treatment with CD40L. A, Anti-Ig induces nuclear κB-binding activity in CD40L-treated B cells in the presence of LY294002. B cells were cultured in medium (M) alone (CD40L(−)), or were stimulated with F(ab′)2 of goat anti-mouse IgM at 15 μg/ml (anti-Ig (αIg)) or with the combination of PMA and ionomycin at 100 and 600 ng/ml, respectively (P/I), for 3 h; B cells were cultured with CD40L/CD8α plus cross-linking anti-CD8 Ab for 48 h (CD40L+/M), or were cultured with CD40L and stimulated with αIg or P/I for the last 3 h of 48 h cultures. B cells were exposed to the PI3K inhibitor, LY294002 at 10 μM (LY), or diluent control DMSO (DM), starting 30 min before addition of anti-Ig or P/I. Nuclei were isolated and extracted as described in Materials and Methods. Nuclear extract protein was tested for binding to a κB site-containing oligonucleotide by EMSA. Binding to an NF-Y site-containing oligonucleotide was used to verify that equal amounts of nuclear extract protein were evaluated. The levels of nuclear κB-binding activity in anti-Ig-treated samples over medium control values after normalization to NF-Y are shown below the corresponding lanes. Results are shown for one of three comparable experiments. B, The composition of NF-κB induced by anti-Ig in CD40L-treated B cells is the same in the presence or absence of PI3K inhibition. Nuclear extract protein samples were obtained from unstimulated (M) or anti-Ig-stimulated (αIg) CD40L-treated B cells in the presence of LY294002 or of control DMSO, and subjected to EMSA, as described above. The identity of anti-Ig-induced κB-binding proteins was evaluated by including Abs that recognize p50, p52, p65, RelB, and c-Rel, as indicated, in the binding reactions to supershift nucleoprotein complexes. Anti-STAT6 Ab was used as a specificity control. One of two comparable experiments is shown.

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The situation was very different for B cells previously treated with CD40L. Although B cell stimulation through CD40 induced nuclear NF-κB early on as expected (36, 40, 41), the level returned to baseline within 2 days (Fig. 1 A and data not shown). Addition of anti-Ig at this time strongly induced NF-κB in B cells treated with LY294002, to a level similar to that observed after anti-Ig stimulation of CD40L-treated B cells exposed to DMSO. Notably, the amount of LY294002 used, 10 μM, was shown in separate dose-titration experiments to completely block anti-Ig-induced phosphorylation of Akt, both with and without prior CD40L treatment (data not shown), Akt being a PI3K substrate in B cells (32). Further, CD40L treatment did not alter the cellular content of PI3K as determined by Western blotting (data not shown).

The nature of this LY294002-resistant, BCR-induced κB-binding activity was further explored through supershift analysis to identify participating Rel-related proteins (Fig. 1,B). The major components of NF-κB induced by anti-Ig stimulation of LY294002-inhibited B cells after CD40L treatment were p50, c-Rel, and p52 along with lesser amounts of immunoreactive p65 and RelB. This same pattern was observed after anti-Ig stimulation of CD40L-treated B cells exposed to DMSO (Fig. 1 B) or after anti-Ig stimulation of naive B cells (data not shown). In these experiments anti-STAT6 was used as a specificity control for the anti-Rel antisera and this reagent did not alter the electrophoretic mobility of κB-binding complexes. These results indicate that in normal B cells BCR engagement induces nuclear NF-κB despite PI3K inhibition when anti-Ig is added after CD40L treatment, and NF-κB induced in this fashion is indistinguishable from NF-κB induced by anti-Ig in control B cells, in direct contrast to the complete failure of anti-Ig to activate NF-κB in naive B cells exposed to LY294002.

NF-κB is retained in the cytosol through binding to inhibitor proteins, such as IκBα, that are eliminated by proteasomal degradation (42). To begin to characterize the mechanism responsible for PI3K-independent, alternate pathway NF-κB activation in normal B cells, we monitored levels of IκBα before and after addition of anti-Ig. Addition of anti-Ig to naive control B cells (CD40L(−)) produced a time-dependent decline in IκBα whereas BCR cross-linking in B cells treated with LY294002 produced little change in IκBα over 90 min (Fig. 2,A), as previously reported (33). However, CD40L treatment for 48 h (CD40L(+)) markedly altered the behavior of IκBα in response to anti-Ig, so that following CD40L, anti-Ig stimulation of LY294002-inhibited B cells produced a decline in cellular IκBα that was as robust as that observed with anti-Ig-stimulated naive B cells, although a little less than that seen with CD40L-treated B cells that were not exposed to LY294002. Similar results were obtained with another, structurally unrelated PI3K inhibitor, wortmannin (Fig. 2 B). As with LY294002, wortmannin completely blocked BCR-induced IκBα degradation in naive B cells (CD40L(−)) over 60 min, as previously reported (33). However, B cell treatment with CD40L for 48 h (CD40L(+)) reinstated the IκBα response to anti-Ig, so that anti-Ig-induced IκBα degradation in CD40L-treated, wortmannin-inhibited B cells was comparable to that observed in naive B cells treated with anti-Ig.

FIGURE 2.

BCR engagement induces IκBα degradation in PI3K-inhibited B cells after treatment with CD40L. A, Anti-Ig (αIg) induces IκBα degradation in CD40L-treated B cells in the presence of the PI3K inhibitor, LY294002. B cells were cultured in medium alone for 3 h (CD40L(−)) or were cultured with CD40L/CD8α plus cross-linking anti-CD8 Ab for 48 h (CD40L(+)); B cells were then stimulated with anti-Ig for the indicated times. B cells were exposed to CHX at 50 μM plus either LY294002 (LY), or diluent control DMSO (DM), starting 30 min before addition of anti-Ig, as indicated. The proteasome inhibitor, lactacystin, was added at 100 μM (Lact) to some samples, as indicated. Whole cell extracts were prepared and Western blotted with anti-IκBα Ab. Blots were stripped and reprobed with anti-actin Ab to verify equal loading of extracted protein. The levels of IκBα after stimulation with anti-Ig, normalized to actin and compared with unstimulated control values, are shown below the corresponding lanes. One of three comparable experiments is shown. B, Anti-Ig induces IκBα degradation in CD40L-treated B cells in the presence of the PI3K inhibitor, wortmannin. B cells were cultured in medium (CD40L(−)) or with CD40L (CD40L(+)) as described above and were then stimulated with anti-Ig for the indicated times. B cells were exposed to CHX at 50 μM plus either wortmannin at 500 nM (Wo), or diluent control DMSO (DM), starting 30 min before addition of anti-Ig, as indicated. Whole cell extracts were prepared and Western blotted as described above. The levels of IκBα after stimulation with anti-Ig, normalized to actin and compared with unstimulated control values, are shown below the corresponding lanes. One of three comparable experiments is shown.

FIGURE 2.

BCR engagement induces IκBα degradation in PI3K-inhibited B cells after treatment with CD40L. A, Anti-Ig (αIg) induces IκBα degradation in CD40L-treated B cells in the presence of the PI3K inhibitor, LY294002. B cells were cultured in medium alone for 3 h (CD40L(−)) or were cultured with CD40L/CD8α plus cross-linking anti-CD8 Ab for 48 h (CD40L(+)); B cells were then stimulated with anti-Ig for the indicated times. B cells were exposed to CHX at 50 μM plus either LY294002 (LY), or diluent control DMSO (DM), starting 30 min before addition of anti-Ig, as indicated. The proteasome inhibitor, lactacystin, was added at 100 μM (Lact) to some samples, as indicated. Whole cell extracts were prepared and Western blotted with anti-IκBα Ab. Blots were stripped and reprobed with anti-actin Ab to verify equal loading of extracted protein. The levels of IκBα after stimulation with anti-Ig, normalized to actin and compared with unstimulated control values, are shown below the corresponding lanes. One of three comparable experiments is shown. B, Anti-Ig induces IκBα degradation in CD40L-treated B cells in the presence of the PI3K inhibitor, wortmannin. B cells were cultured in medium (CD40L(−)) or with CD40L (CD40L(+)) as described above and were then stimulated with anti-Ig for the indicated times. B cells were exposed to CHX at 50 μM plus either wortmannin at 500 nM (Wo), or diluent control DMSO (DM), starting 30 min before addition of anti-Ig, as indicated. Whole cell extracts were prepared and Western blotted as described above. The levels of IκBα after stimulation with anti-Ig, normalized to actin and compared with unstimulated control values, are shown below the corresponding lanes. One of three comparable experiments is shown.

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To verify that IκBα loss in the PI3K-independent, CD40L-induced BCR signaling pathway involves proteasomal degradation, we blocked proteasomal function with the inhibitor, lactacystin. In preliminary titration experiments we found that lactacystin at 100 μM completely blocked anti-Ig-induced IκBα degradation in naive B cells, although some investigators have reported using lesser amounts (43). As shown in Fig. 2 A, lactacystin blocked anti-Ig-induced IκBα loss similarly in naive B cells and in CD40L-treated, LY294002-inhibited B cells. Further, and as expected from the preceding, lactacystin blocked anti-Ig-induced NF-κB activation similarly in both CD40L-treated and untreated B cells (data not shown).

Altogether these results indicate that an alternate signaling pathway for BCR-induced NF-κB activation is established in normal B cells by CD40L treatment that bypasses the need for PI3K. NF-κB induced by this pathway consists of the same Rel components triggered by anti-Ig in naive B cells, and results from IκBα degradation via proteasomal activity, just like inducible NF-κB in naive B cells.

IκBα is targeted for proteasomal degradation by inducible phosphorylation. The canonical pathway responsible for this process begins with phosphorylation and activation of the IKK complex which phosphorylates IκBα on serines 32 and 36. To explore the mechanism responsible for IκBα degradation via the PI3K-independent alternate BCR signaling pathway in normal B cells, we examined IκBα serine 32/36 phosphorylation by Western blotting using phosphoserine 32/36-specific Abs.

Anti-Ig stimulation produced substantial immunoreactive IκBα serine 32/36 phosphorylation in naive B cells (CD40L(−)) within 5 min; IκBα phosphorylation extended to at least 30 min in a reproducibly biphasic fashion as might be expected from previous results on IKK activation (Fig. 3 A) (19). This anti-Ig-induced IκBα phosphorylation was completely blocked in naive B cells by LY294002 for at least 30 min. CD40L treatment for 48 h (CD40L(+)) did not alter the absent baseline level of IκBα phosphorylation. Anti-Ig stimulation after CD40L treatment again stimulated immunoreactive IκBα serine 32/36 phosphorylation in control (DMSO-treated) B cells that was much greater at 5 min than at later time points. Although the early appearing phospho-IκBα produced by anti-Ig stimulation of CD40L-treated B cells was markedly reduced by PI3K inhibition, anti-Ig-induced IκBα phosphorylation at later time points was similar to that observed in the absence of LY294002 and was clearly increased over that in CD40L(−)/LY294002 B cells. The same pattern was observed when IκBα phosphorylation was monitored with a different, anti-phosphoserine 32 IκBα reagent (data not shown).

FIGURE 3.

BCR engagement induces IκBα phosphorylation in CD40L-treated, PI3K-inhibited B cells. A, BCR engagement induces immunoreactive IκBα Ser32/36 phosphorylation in CD40L-treated, LY294002-inhibited B cells. B cells were cultured in medium alone for 3 h (CD40L(−)) or were cultured with CD40L for 48 h (CD40L(+)) and then stimulated for the indicated times with anti-Ig at 15 μg/ml (α-Ig) in the presence of LY294002 at 10 μM (LY) or diluent control DMSO (DM) starting 30 min before addition of anti-Ig. Whole cell extracts were prepared and Western blotted as described in Materials and Methods with anti-phospho-IκBα (Ser32/36) Ab. Blots were stripped and reprobed with anti-IκBα and anti-actin Abs. One of three comparable experiments is shown. The levels of phospho-IκBα after stimulation with anti-Ig, normalized to IκBα and compared with unstimulated control values, are shown below the corresponding lanes. B, Lactacystin enhances BCR-induced IκBα Ser32/36 phosphorylation in CD40L-treated, LY294002-inhibited B cells. B cells were cultured with CD40L for 48 h (CD40L(+)) and then stimulated for the indicated times with anti-Ig in the presence of LY294002 or control diluent DMSO starting 30 min before addition of anti-Ig. Lactacystin at 100 μM was added 30 min before anti-Ig, as indicated. Whole cell extracts were prepared and Western blotted with anti-phospho-IκBα (Ser32/36) Ab. Blots were stripped and reprobed with anti-IκBα and anti-actin Abs. The levels of phospho-IκBα after stimulation with anti-Ig, normalized to IκBα and compared with unstimulated control values, are shown below the corresponding lanes. One of three comparable experiments is shown.

FIGURE 3.

BCR engagement induces IκBα phosphorylation in CD40L-treated, PI3K-inhibited B cells. A, BCR engagement induces immunoreactive IκBα Ser32/36 phosphorylation in CD40L-treated, LY294002-inhibited B cells. B cells were cultured in medium alone for 3 h (CD40L(−)) or were cultured with CD40L for 48 h (CD40L(+)) and then stimulated for the indicated times with anti-Ig at 15 μg/ml (α-Ig) in the presence of LY294002 at 10 μM (LY) or diluent control DMSO (DM) starting 30 min before addition of anti-Ig. Whole cell extracts were prepared and Western blotted as described in Materials and Methods with anti-phospho-IκBα (Ser32/36) Ab. Blots were stripped and reprobed with anti-IκBα and anti-actin Abs. One of three comparable experiments is shown. The levels of phospho-IκBα after stimulation with anti-Ig, normalized to IκBα and compared with unstimulated control values, are shown below the corresponding lanes. B, Lactacystin enhances BCR-induced IκBα Ser32/36 phosphorylation in CD40L-treated, LY294002-inhibited B cells. B cells were cultured with CD40L for 48 h (CD40L(+)) and then stimulated for the indicated times with anti-Ig in the presence of LY294002 or control diluent DMSO starting 30 min before addition of anti-Ig. Lactacystin at 100 μM was added 30 min before anti-Ig, as indicated. Whole cell extracts were prepared and Western blotted with anti-phospho-IκBα (Ser32/36) Ab. Blots were stripped and reprobed with anti-IκBα and anti-actin Abs. The levels of phospho-IκBα after stimulation with anti-Ig, normalized to IκBα and compared with unstimulated control values, are shown below the corresponding lanes. One of three comparable experiments is shown.

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To evaluate the possibility that detection of IκBα phosphorylated on serines 32 and 36 might have been obscured by rapid proteasomal degradation, we re-evaluated induction of immunoreactive phosphoserine 32/36 IκBα in the presence of the proteasomal inhibitor, lactacystin. In CD40L-treated B cells, lactacystin produced an increase in immunoreactive phosphoserine 32/36 stimulated by anti-Ig whether or not LY294002 was present (Fig. 3 B). Thus, phospho-IκBα induced in CD40L-treated B cells that is resistant to LY294002 is proteasomally degraded and rescued by lactacystin, although the level of phospho-IκBα, even in the presence of lactacystin, remained less than that apparent in CD40L-treated, non-PI3K-inhibited control B cells.

The IKK complex consists of two kinase moieties, IKKα and IKKβ, that are activated by phosphorylation (24). To explore the role of IKK in the PI3K-independent alternate BCR signaling pathway, we examined IKKα phosphorylation (Ser180) and IKKβ phosphorylation (Ser181) by Western blotting with specific antisera (Fig. 4). Anti-Ig stimulation produced substantial immunoreactive IKKβ phosphorylation in naive B cells (CD40L(−)) within 5 min that continued for at least 30 min, whereas phospho-IKKα was constitutively expressed. These results are consistent with previous work indicating that IKKβ activation is primarily responsible for NF-κB induction in response to anti-IgM (44). Anti-Ig-induced IKKβ phosphorylation was completely blocked in naive B cells by LY294002. After CD40L treatment for 48 h (CD40L(+)), anti-Ig again stimulated robust phosphorylation of IKKβ in control (DMSO-treated) B cells over a background that was unchanged as a result of CD40L treatment. Most importantly, after CD40L treatment, LY294002 now failed to block anti-Ig-induced IKKβ phosphorylation; that is, the pathway leading from BCR to IKKβ phosphorylation had become PI3K-independent as a result of prior CD40 engagement. This pattern of LY294002 resistance for BCR-induced phospho-IKKβ following CD40L treatment recapitulated the results obtained for NF-κB activation and IκBα degradation, and suggested that IKKβ activation is responsible for these latter outcomes.

FIGURE 4.

BCR engagement induces IKKβ phosphorylation in CD40L-treated B cells despite inhibition of PI3K. B cells were cultured in medium alone for 3 h (CD40L(−)) or were cultured with CD40L for 48 h (CD40L(+)) and then stimulated for the indicated times with anti-Ig at 15 μg/ml (αIg) in the presence of LY294002 at 10 μM (LY) or diluent control DMSO (DM) starting 30 min before addition of anti-Ig. Whole cell extracts were prepared and Western blotted as described in Materials and Methods with anti-phospho-IKKα/IKKβ Ab. Blots were stripped and reprobed with IKKα-specific and IKKβ-specific Abs. One of five comparable experiments is shown.

FIGURE 4.

BCR engagement induces IKKβ phosphorylation in CD40L-treated B cells despite inhibition of PI3K. B cells were cultured in medium alone for 3 h (CD40L(−)) or were cultured with CD40L for 48 h (CD40L(+)) and then stimulated for the indicated times with anti-Ig at 15 μg/ml (αIg) in the presence of LY294002 at 10 μM (LY) or diluent control DMSO (DM) starting 30 min before addition of anti-Ig. Whole cell extracts were prepared and Western blotted as described in Materials and Methods with anti-phospho-IKKα/IKKβ Ab. Blots were stripped and reprobed with IKKα-specific and IKKβ-specific Abs. One of five comparable experiments is shown.

Close modal

To determine whether IKKβ activation is responsible for IκBα degradation/NF-κB induction in LY294002-treated B cells after CD40L stimulation, we used the recently reported novel IKKβ inhibitor, jesterone dimer (JD; kindly provided by Dr. J. A. Porco, Boston University, Boston, MA) (37). As reported by Liang et al. (37), JD is a synthetic derivative of the natural fungal metabolite, jesterone, which produces a high molecular complex of IKK family members that blocks IκBα phosphorylation and NF-κB activation. We initially confirmed that JD is active in naive murine B cells by determining the optimal concentration needed to interfere with IκBα phosphorylation and degradation. Anti-Ig-induced IκBα degradation was blocked by JD at 10 μM or higher (Fig. 5,A), and the same was true of IκBα phosphorylation (data not shown). Using JD at 20 μM, we then evaluated the effect of IKK inhibition on BCR-induced IκBα degradation after B cell treatment with CD40L treatment. In the absence of JD (JD(−)), anti-Ig induced IκBα degradation irrespective of the presence of LY294002 (Fig. 5,B), as expected from the results shown in Fig. 2, although degradation was a little less with LY294002 than without. However, when JD was present (JD(+)), IκBα degradation was completely blocked, both in control (DMSO-treated) and LY294002-treated B cells, indicating that IKK activity is required in both situations.

FIGURE 5.

IKK activation is required for BCR-induced IκBα degradation in the presence of LY294002 after CD40L stimulation. A, Inhibition of IKK activity with JD blocks BCR-induced IκBα degradation. Naive B cells were cultured with JD at the indicated doses for 1.5 h, and then JD plus CHX for 30 min, followed by stimulation with anti-Ig (αIg) for 60 min, as indicated. Whole cell extracts were prepared and Western blotted with anti-IκBα Ab. Blots were stripped and reprobed with anti-actin Ab. The levels of IκBα after stimulation with anti-Ig and/or treatment with JD, normalized to actin and compared with the unstimulated control value, are shown below the corresponding lanes. One of two comparable experiments is shown. B, Inhibition of IKK activity with JD blocks BCR-induced IκBα degradation in CD40L-treated, PI3K-inhibited B cells. B cells were cultured with CD40L for 48 h and then exposed to JD at 20 μM for 1.5 h followed by either JD plus LY294002 plus CHX (JD(+)/LY) or JD plus control diluent DMSO plus CHX (JD(+)/DM) for 30 min; alternatively CD40L-treated B cells were exposed to medium for 1.5 h followed by either LY294002 plus CHX (JD(−)/LY) or control diluent DMSO plus CHX (JD(−)/DM) for 30 min. B cells were then stimulated with anti-Ig (αIg) for 60 min, as indicated. Whole cell extracts were prepared and Western blotted with anti-IκBα Ab. Blots were stripped and reprobed with anti-actin Ab. The levels of IκBα after stimulation with anti-Ig and/or treatment with LY, normalized to actin and compared with JD(−) or JD(+) unstimulated control values, are shown below the corresponding lanes. One of two comparable experiments is shown.

FIGURE 5.

IKK activation is required for BCR-induced IκBα degradation in the presence of LY294002 after CD40L stimulation. A, Inhibition of IKK activity with JD blocks BCR-induced IκBα degradation. Naive B cells were cultured with JD at the indicated doses for 1.5 h, and then JD plus CHX for 30 min, followed by stimulation with anti-Ig (αIg) for 60 min, as indicated. Whole cell extracts were prepared and Western blotted with anti-IκBα Ab. Blots were stripped and reprobed with anti-actin Ab. The levels of IκBα after stimulation with anti-Ig and/or treatment with JD, normalized to actin and compared with the unstimulated control value, are shown below the corresponding lanes. One of two comparable experiments is shown. B, Inhibition of IKK activity with JD blocks BCR-induced IκBα degradation in CD40L-treated, PI3K-inhibited B cells. B cells were cultured with CD40L for 48 h and then exposed to JD at 20 μM for 1.5 h followed by either JD plus LY294002 plus CHX (JD(+)/LY) or JD plus control diluent DMSO plus CHX (JD(+)/DM) for 30 min; alternatively CD40L-treated B cells were exposed to medium for 1.5 h followed by either LY294002 plus CHX (JD(−)/LY) or control diluent DMSO plus CHX (JD(−)/DM) for 30 min. B cells were then stimulated with anti-Ig (αIg) for 60 min, as indicated. Whole cell extracts were prepared and Western blotted with anti-IκBα Ab. Blots were stripped and reprobed with anti-actin Ab. The levels of IκBα after stimulation with anti-Ig and/or treatment with LY, normalized to actin and compared with JD(−) or JD(+) unstimulated control values, are shown below the corresponding lanes. One of two comparable experiments is shown.

Close modal

These results strongly suggest that the CD40L-induced, PI3K-independent alternate pathway for BCR signaling present in normal B cells eventuates in IKKβ phosphorylation and activation that is responsible for subsequent IκBα degradation.

The extent to which the CD40L-induced, PI3K-independent, alternate pathway for BCR signaling diverges from the canonical signalosome pathway remains undefined. To begin to address this issue, we examined general and specific protein tyrosine phosphorylation stimulated by anti-Ig in naive and CD40L-treated B cells, in the presence and absence of LY294002. General tyrosine phosphorylation was evaluated by Western blotting whole cell extracts with phosphotyrosine-specific Ab. Anti-Ig stimulation of naive B cells resulted in tyrosine phosphorylation of a variety of proteins as has been reported previously (45, 46), which was little changed by the presence of LY294002, as might be expected because PI3K lies downstream of src-like kinase activity in the current paradigm for BCR signal propagation (Fig. 6 A). Following CD40L treatment, anti-Ig again stimulated tyrosine phosphorylation of a variety of proteins, and although the magnitude of phosphoprotein staining appeared somewhat increased in comparison to naive B cells, the distribution of phosphorylated proteins appeared largely unchanged. As with naive B cells, the presence of LY294002 had little effect. Thus, we detected no obvious divergence in tyrosine-phosphorylated proteins that might correlate with initiation of the alternate pathway for BCR signaling.

FIGURE 6.

The alternate pathway for BCR signaling does not substantially impact general tyrosine phosphorylation or phosphorylation of BLNK or PLC. A, BCR-induced tyrosine phosphorylation is not substantially affected by B cell treatment with CD40L or LY294002. B cells were cultured in medium alone for 3 h (CD40L(−)) or were cultured with CD40L for 48 h (CD40L(+)) and then stimulated for the indicated times with anti-Ig in the presence of LY294002 or diluent control DMSO starting 30 min before addition of anti-Ig. Whole cell extracts were prepared and Western blotted with anti-phosphotyrosine Ab. Blots were stripped and reprobed with anti-actin Ab. One of three comparable experiments is shown. B, BCR-induced phosphorylation of BLNK and PLCγ2 are not enhanced by B cell treatment with CD40L or blocked by treatment with LY294002. B cells were treated with CD40L, LY294002 (LY) and DMSO (DM) as described above, and were then stimulated with anti-Ig (αIg) for 5 min, as indicated. Equal aliquots of protein lysates were subjected to immunoprecipitation as described in Materials and Methods and immune complexes were Western blotted with anti-phosphotyrosine Ab. Blots were stripped and reprobed with anti-BLNK and anti-PLCγ2. One of three comparable experiments is shown.

FIGURE 6.

The alternate pathway for BCR signaling does not substantially impact general tyrosine phosphorylation or phosphorylation of BLNK or PLC. A, BCR-induced tyrosine phosphorylation is not substantially affected by B cell treatment with CD40L or LY294002. B cells were cultured in medium alone for 3 h (CD40L(−)) or were cultured with CD40L for 48 h (CD40L(+)) and then stimulated for the indicated times with anti-Ig in the presence of LY294002 or diluent control DMSO starting 30 min before addition of anti-Ig. Whole cell extracts were prepared and Western blotted with anti-phosphotyrosine Ab. Blots were stripped and reprobed with anti-actin Ab. One of three comparable experiments is shown. B, BCR-induced phosphorylation of BLNK and PLCγ2 are not enhanced by B cell treatment with CD40L or blocked by treatment with LY294002. B cells were treated with CD40L, LY294002 (LY) and DMSO (DM) as described above, and were then stimulated with anti-Ig (αIg) for 5 min, as indicated. Equal aliquots of protein lysates were subjected to immunoprecipitation as described in Materials and Methods and immune complexes were Western blotted with anti-phosphotyrosine Ab. Blots were stripped and reprobed with anti-BLNK and anti-PLCγ2. One of three comparable experiments is shown.

Close modal

To address the early tyrosine phosphorylation cascade in more detail, we examined phosphorylation of two key signalosome elements, BLNK, and PLCγ2, by immunoprecipitation followed by Western blotting. BLNK acts as a key adaptor protein in BCR signaling. Anti-Ig stimulation produced substantial BLNK phosphorylation in naive B cells that was independent of PI3K inhibition (Fig. 6 B), as might be expected because the principal BLNK kinase is Syk (47). Following CD40L treatment, anti-Ig again stimulated substantial BLNK phosphorylation which was again relatively resistant to PI3K inhibition with LY294002. This result suggests that whatever the nature of the alternate pathway may be, it does not appear to interfere with operation of the canonical pathway.

PLC plays a central role in signal propagation by producing second messenger molecules responsible for activating PKC and for raising intracellular Ca2+. Anti-Ig stimulation produced PLCγ2 phosphorylation in naive B cells; surprisingly, PLCγ2 phosphorylation was independent of PI3K inhibition (Fig. 6 B), even though the generally accepted paradigm for PLCγ2 activation involves contributions from Syk and Btk, the latter being sensitive to PI3K activity (48, 49, 50). Following CD40L treatment, anti-Ig again stimulated PLCγ2 phosphorylation which was again resistant to PI3K inhibition with LY294002. This finding supports the results obtained with BLNK in suggesting that the alternate pathway does not interfere with the canonical pathway for BCR signaling. However, at least some elements of the canonical pathway may be involved in mediating the alternate pathway for BCR signaling, inasmuch as inhibition of Syk with piceatannol blocked anti-Ig-induced IκBα degradation in CD40L-treated B cells (data not shown).

The uninterrupted BCR-triggered phosphorylation of the downstream signalosome element, PLCγ2, raised the possibility that the alternate pathway relies on PLC activity and simply bypasses or substitutes for Btk/PI3K activation. This possibility is reinforced by previous results in PLCγ2-deficient mice and PLC-inhibited B cells indicating that PLCγ2 is required for anti-Ig-induced activation of NF-κB (51). To test the role of PLCγ2 directly, we made use of the PLC inhibitor, U73122. Previous work indicated that U73122 treatment of primary B cells blocked IKK activity (51). As expected, anti-Ig-induced IκBα degradation was completely blocked by U73122 at 1 μM (52) in naive B cells (Fig. 7). However, following CD40L treatment, IκBα degradation was induced by anti-Ig despite the presence of U73122, much like the situation observed with LY294002 and wortmannin (Fig. 2). In separate control titration experiments we showed that the dose of U73122 used, 1 μM, completely blocked the anti-Ig-induced increase in intracellular Ca2+ in both CD40L-treated and untreated B cells; further, CD40L treatment did not alter the cellular content of PLCγ2 as determined by Western blotting (data not shown). Thus, the pathway leading from BCR to IκBα degradation became PLCγ2-independent (as well as PI3K-independent) as a result of prior CD40 engagement.

FIGURE 7.

The CD40L-induced alternate pathway for BCR signaling bypasses PLCγ2. B cells were treated as described in the legend to Fig. 2 B, with the exception that PLCγ2 was inhibited with U73122 at 1 μM (U73). Whole cell extracts were prepared and Western blotted with anti-IκBα Ab. Blots were stripped and reprobed with anti-actin Ab. The levels of IκBα after stimulation with anti-Ig, normalized to actin and compared with unstimulated control values, are shown below the corresponding lanes. One of three comparable experiments is shown.

FIGURE 7.

The CD40L-induced alternate pathway for BCR signaling bypasses PLCγ2. B cells were treated as described in the legend to Fig. 2 B, with the exception that PLCγ2 was inhibited with U73122 at 1 μM (U73). Whole cell extracts were prepared and Western blotted with anti-IκBα Ab. Blots were stripped and reprobed with anti-actin Ab. The levels of IκBα after stimulation with anti-Ig, normalized to actin and compared with unstimulated control values, are shown below the corresponding lanes. One of three comparable experiments is shown.

Close modal

We previously demonstrated that an alternate pathway for B cell signaling exists in xid B cells–we showed that this pathway is established by CD40L treatment and circumvents the need for Btk in BCR-induced NF-κB activation (28). The present work greatly expands on these initial observations, most importantly by showing that the alternate pathway is not idiosyncratic to xid B cells but exists in normal B cells as well. Further, we have shown here that the alternate BCR signaling pathway operates without the need for PI3K or PLCγ2, and thus bypasses both early (Btk and PI3K) as well as late (PLCγ2) signalosome elements. At the same time, IKKβ remains a central and required element for proteasomal IκBα degradation/nuclear NF-κB activation in this pathway as well as in the conventional pathway. Thus, the alternate pathway appears to circumvent the signalosome but terminate in IKK phosphorylation and activation.

Creation of the alternate pathway does not appear to interfere with the conventional pathway for BCR signaling, which remains intact in terms of general protein tyrosine phosphorylation as well as phosphorylation of specific conventional pathway mediators. This may explain a minor aspect of our results. Typically, IκBα degradation produced by anti-Ig stimulation of CD40L-treated, PI3K-inhibited B cells was not as great as that produced by anti-Ig stimulation of B cells treated with CD40L in the absence of an inhibitor. Along the same lines, IκBα degradation produced by anti-Ig stimulation of CD40L-treated B cells was typically greater than that produced by anti-Ig stimulation of naive B cells (see Figs. 2,A, 5,B, and 7). The likely explanation is that CD40L treatment establishes an alternate pathway that operates in parallel to, not in conflict with, the classical pathway. Thus, two pathways, the classical and the alternate, converge to produce IκBα degradation after anti-Ig stimulation of CD40L-treated B cells, but only a single pathway is triggered by anti-Ig in either naive B cells (the classical pathway) or CD40L-treated, PI3K-inhibited (or PLCγ-inhibited) B cells (the alternate pathway).

Many of the functional results reported herein rely on the use of metabolic inhibitors. The use of inhibitors in these experiments provided the opportunity to evaluate the existence of the alternate pathway in normal, as opposed to mutant, B cells by interfering with function in real time. B cells that express a mutant mediator are highly likely to express other gene products at abnormal levels, as already shown for xid and Btk−/− B cells (30), and it is impossible to know whether one or more of such abnormally expressed molecules might contribute to, or be responsible for, the alternate pathway in mutant B cells. In particular, the use of PI3K inhibitors is advantageous as compared with the use of B cells obtained from PI3K-deficient animals, because PI3K-deficient B cells do not proliferate normally in response to CD40 engagement (7, 39), suggesting that other aspects of CD40 signaling may be deranged as well, in which case it would be impossible to distinguish failure to establish the alternate pathway from an alternate pathway that does not bypass PI3K. Confidence in the results reported here is enhanced by the fact that two unrelated inhibitors produced similar results with respect to the role of PI3K. Further, LY294002 was shown to be specific in its downstream effects, blocking Akt, but not IKKβ, activation in CD40L-treated B cells, lending credence to the idea that these inhibitors do not globally interfere with all signaling mediators.

Although inhibition of PI3K did not block the alternate pathway for BCR signaling, surprisingly, it also did not interfere with BCR-induced phosphorylation of PLCγ2, either before or after B cell treatment with CD40L. Because PLCγ2 activation relies in part on Btk (3), activation of which is enhanced by PI3K activity (48, 49, 50), this latter result seems counterintuitive. Very recently, however, it was reported that BCR engagement produces normal PLCγ2 phosphorylation in B cells obtained from p110δ−/− (PI3K−/−) mice, although Ca2+ mobilization in the latter was only 25% of normal (11). Thus, normal tyrosine phosphorylation of PLCγ2 does not necessarily imply full PLCγ2 activation. Our results with U73122 confirm previous work indicating that PLCγ2 is required for BCR-induced NF-κB activation in naive B cells (51), even as we showed that PLCγ2 is not required for operation of the CD40L-induced alternate pathway for BCR signaling.

Both the alternate pathway and the conventional pathway produce IKKβ phosphorylation, IκBα phosphorylation, IκBα degradation, and NF-κB activation. Further, blocking IKKβ interfered with subsequent downstream events. However, although anti-Ig induced phospho-IκBα in the face of LY294002 after CD40L treatment that was proteasomally degraded, the level of phospho-IκBα was clearly less than that observed in the absence of PI3K inhibition. The level of phospho-IκBα detected may relate, in part, to the kinetics of IκBα turnover. However, in UV-induced NF-κB activation, IκBα degradation through the proteasome proceeds with minimal IκBα serine 32/36 phosphorylation (53, 54). At the same time, a role for IKK has been implicated because inhibition of IKK, or overexpression of mutant, inactive IKKβ, blocked UV-induced NF-κB activation (55). Thus, the level of IκBα phosphorylation detected by Western blotting may not directly reflect the importance of IκBα phosphorylation in mediating subsequent proteasomal degradation and NF-κB activation.

This raises the question of the precise molecular structure of the alternate pathway–how BCR is connected to IKK, and how IKK is connected IκBα. A number of molecules appear capable of phosphorylating IKKβ, but it is unknown which, if any, of these might participate in bridging BCR and IKKβ under normal circumstances or in the alternate pathway. Further, the role of putative adaptor/scaffolding proteins, such as Bcl10, MALT1, and CARMA1 (56, 57, 58, 59, 60), remains uncertain, although Bcl10 has been shown to be required for BCR-mediated induction of NF-κB (57).

Having shown that the alternate pathway, in which prior CD40 engagement alters subsequent BCR signaling, characterizes normal B cells, it is natural to consider the relevance of this sequence of events for normal immune responses. Although the classical paradigm holds that a restricted B cell population is initially activated by Ag and is only then stimulated by peptide-specific T cells, in some cases naive B cells are first stimulated through CD40 (61). Such bystander B cell activation has been documented in several ways, most notably through B cell responsiveness to a nonlinked, nonimmunogenic protein as a result of immunogenic, T-dependent Ag administration (62). More recently, Rajewsky and colleagues (63) documented germinal center formation in the absence of BCR expression in a mutant mouse strain and in three additional strains of mice expressing nonautoreactive, transgenic, BCRs in which B cells were never exposed to Ag. Thus, naive B cells in certain situations or in special locations may be stimulated physiologically through CD40 before BCR.

As described here and elsewhere, the alternate pathway is evident after CD40L treatment for 24–48 h (28). This may be an overestimate of the time required to establish this pathway, because CD40L itself induces NF-κB (40, 41) so the alternate pathway for BCR signaling to IκBα/NF-κB can only be detected after CD40-triggered events have returned to baseline. Still, the time required for generation of the alternate pathway raises the question of whether contact between T cells and B cells can actually last that long. Although definitive data on this issue is not available, what data there is suggests that B cells are influenced by CD40 signaling over long periods of time. Thus, administration of anti-CD40L Ab disrupts B cell germinal center structures and interferes with production of high affinity Ab when administered either soon, or many days, after germinal center formation, even 16–20 days after initiation of an immune response (64, 65, 66). Further, CD40L expression on activated T cells is prolonged, and interference with CD40L at various timepoints over a period of days affects B cell terminal differentiation (67). Thus, the influence of CD40L in creating a new, and apparently less stringent, alternate pathway in vitro, that bridges BCR and IKKβ without the need for Btk, PI3K, or PLCγ, may well represent events that occur commonly in vivo.

The authors thank Dr. J. A. Porco for kindly providing JD.

The authors have no financial conflict of interest.

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 Public Health Service Grant AI40181 awarded by the National Institutes of Health.

4

Abbreviations used in this paper: anti-Ig, F(ab′)2 of anti-IgM Ab; BLNK, B cell linker; Btk, Bruton’s tyrosine kinase; CHX, cycloheximide; IKK, IκB kinase; JD, jesterone dimer; PH, pleckstrin homology; PKC, protein kinase C; phospho-IκBα, phosphorylated IκBα; PLC, phospholipase C; SH, Src homology.

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