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Cutting Edge: CD40 Engagement Eliminates the Need for Bruton’s Tyrosine Kinase in B Cell Receptor Signaling for NF-B¹

Takuya Mizuno^*, and Thomas L. Rothstein^2,*,,

Departments of ^* Medicine and Microbiology, Boston University School of Medicine, and Immunobiology Unit, Evans Memorial Department of Clinical Research, Boston University Medical Center, Boston, MA 02118

	Abstract

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The Tec kinase Bruton’s tyrosine kinase (Btk) represents a key intermediary for B cell receptor (BCR) signaling. Btk mutation produces B cell deficiency in mice with X-linked immunodeficiency (xid), and surface Ig-mediated responses of mature B cells are seriously deranged. The central role that Btk plays in directing downstream events produced by BCR engagement is demonstrated by the complete failure of NF-B induction and cellular proliferation following anti-Ig treatment of B cells obtained from xid mice. In this study, we report that the block in BCR signaling produced by Btk mutation is reversed by CD40 engagement. Prior treatment with CD40 ligand normalized subsequent responses of xid B cells to BCR cross-linking, so that typical outcomes of BCR signaling such as NF-B activation and cell cycle progression occurred in a Btk-independent fashion. These results demonstrate that a specific genetic lesion interrupting BCR-mediated intracellular signaling is circumvented through stimulation of CD40.

	Introduction

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Bruton’s tyrosine kinase (Btk)³ plays a key role in B cell physiology (reviewed in Ref. 1). Mutation of Btk affects B cell development, leading to diminished numbers of mature B cells in the periphery. This is particularly evident in the human disease, X-linked agammaglobulinemia, which is characterized by the virtual absence of mature B cells resulting in marked deficiency of serum Ab (2, 3). The corresponding murine dyscrasia, X-linked immunodeficiency (xid), is characterized by a less pronounced developmental block resulting in a reduction (to about half of normal) but not elimination of mature B cells (4, 5). Although present, xid B cells are functionally abnormal in that B cell receptor (BCR) signaling is severely compromised, and typical outcomes of surface Ig (sIg) cross-linking such as induction of nuclear NF-B and progression through cell cycle do not occur (6, 7, 8, 9). Even when sufficient Btk is provided to xid to normalize B cell development, BCR signaling is still impaired (10). Thus, Btk plays a critical and perhaps limiting role in the propagation of sIg-induced intracellular signaling.

The susceptibility of B cells to Fas-mediated apoptosis is modulated by specific receptor signaling, and is opposed by BCR engagement which produces a state of Fas resistance, even in otherwise Fas-sensitive CD40L-stimulated targets (reviewed in Ref. 11). The mitogenic combination of PMA plus ionomycin (P/I) also protects CD40 ligand (CD40L)-stimulated B cells from Fas killing (12). The mechanism by which sIg signaling establishes Fas resistance has been the subject of much investigation. Early work demonstrated that anti-Ig treatment produced Fas resistance only after several hours of exposure, and that new gene expression and new protein synthesis were required (12, 13). The implication that Fas resistance depends on transcriptional activation raised the possibility of a role for NF-B, which in other situations has been shown to be antiapoptotic and/or to up-regulate expression of antiapoptotic molecules (reviewed in Ref. 14). In recent work, we found that inhibition of NF-B induction blocked the ability of anti-Ig to produce Fas resistance (15). Consistent with this, induction of two NF-B-dependent antiapoptotic molecules implicated in sIg-induced Fas resistance was also blocked by NF-B inhibition (15).

The dependence of many sIg-triggered downstream events on Btk suggested that this kinase would play a role in mediating the induction of Fas resistance. However, we found that the ability of anti-Ig to produce Fas resistance was not substantially affected by mutation or deletion of Btk (16). In light of recent reports indicating that Btk is absolutely required for NF-B activation (8, 9), these two results, indicating that induction of Fas resistance depends on NF-B but occurs independently of Btk, would seem to be at odds with one another. Notably, in previous work, the demonstration of Fas resistance involved the addition of anti-Ig to CD40L-stimulated B cells to create conditions in which sIg engagement reverses, rather than prevents, Fas sensitivity, thereby presenting the most stringent test for modulation of Fas sensitivity (11). This suggested the possibility that CD40L stimulation might provide the means to circumvent the block in sIg signaling produced by Btk mutation, such that even in xid B cells sIg cross-linking would induce NF-B, thereby fulfilling the previously established parameters for sIg-induced Fas resistance, consisting of NF-B dependence but Btk independence. The present study was designed to explore the possibility that CD40L treatment allows sIg signaling to successfully bypass Btk and propagate otherwise blocked downstream events.

	Materials and Methods

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Animals

CBA/CaHN-Btkxid/J mice (xid) and CBA/CaJ (wild-type (wt) control) mice were obtained from The Jackson Laboratory (Bar Harbor, ME).

Primary B cell culture

Spleen cells from 2- to 3-mo-old mice were depleted of T cells, RBC, and nonviable cells as previously described (17). The resulting B cells were cultured in RPMI 1640 medium (BioWhittaker, Walkersville, MD) supplemented with 5% FBS (Sigma-Aldrich, St. Louis, MO), 10 mM HEPES (Calbiochem-Novabiochem, San Diego, CA), 5 x 10^-5 M 2-ME (Sigma-Aldrich), 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM glutamine.

EMSA

Nuclear extracts were prepared as described previously (18). Briefly, nuclei were collected following hypotonic lysis of stimulated cells and extracted for 60 min on ice in a high salt buffer containing 430 mM NaCl and protease inhibitors. Binding reactions containing 1.0 µg of nuclear protein, 0.5 µg of poly(dI-dC) (Amersham Biosciences, Piscataway, NJ), and radiolabeled B site-containing oligonucleotide probe (18, 19) were incubated for 30 min at room temperature. NF-Y binding (Santa Cruz Biotechnology, Santa Cruz, CA) was used to verify that equal amounts of nuclear extract protein were present in each sample (20). For supershift analysis, nuclear extracts were preincubated for 1 h on ice with Rel-specific Abs (generously provided by Dr. N. Rice (National Cancer Institute, Frederick, MD)) before ³²P-labeled probe was added. Affinity-purified rabbit polyclonal STAT6-specific Ab (Santa Cruz Biotechnology) was used as a specificity control for supershift analysis.

Proliferation assay

Splenic B cells (2 x 10⁵ in 0.2 ml) were cultured for 48 h in flat-bottom microtiter walls in quadruplicate, as previously described (21). Tritium incorporation was assessed after exposure to 0.5 µCi of [³H]thymidine (20 Ci/mmol; New England Nuclear, Boston, MA) during the last 6 h of culture.

Western blotting

Cytoplasmic protein was extracted from cell pellets with 1% Nonidet P-40 lysis buffer containing protease inhibitors, and equal amounts (25–50 µg) were subjected to SDS-PAGE followed by immunoblotting with rabbit primary and HRP-labeled anti-rabbit secondary Abs, as described (22). Membranes were developed using SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, IL). To verify that equal amounts of protein were added to each lane, blots were stripped and reprobed with anti-p38 Ab (Santa Cruz Biotechnology) for IB degradation analysis and with anti-actin Ab (Sigma-Aldrich) for total cellular NF-B analysis. For evaluation of IB degradation, cycloheximide (50 µM) was added to B cell cultures 30 min before addition of anti-IgM, as described (9). IB-specific Ab and Abs that recognize p65, RelB, and c-Rel were obtained from Santa Cruz Biotechnology; Abs specific for p50 and p52 were generously provided by Dr. N. Rice.

Reagents

Affinity-purified F(ab')₂ polyclonal goat anti-mouse IgM (anti-Ig) were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). Soluble rCD40L was obtained from transfected J558L cells that secrete a chimeric CD40L/CD8 fusion protein (23) and was dialyzed as previously described (24). Anti-CD8 Ab was obtained from the supernatant of 53-6-72 hybridoma cells and dialyzed similarly. PMA, ionomycin, and LPS were obtained from Sigma-Aldrich.

	Results

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BCR engagement activates NF-B in xid B cells after stimulation by CD40L

To explore the possibility that sIg signaling requirements are altered by CD40 engagement, we obtained purified splenic B cells from xid and wt control mice and then examined nuclear extracts prepared before and after B cell stimulation with CD40L and/or anti-Ig for expression of NF-B by EMSA. Results are shown in Fig. 1.

View larger version (93K): [in this window] [in a new window]   FIGURE 1. BCR engagement activates NF-B in xid B cells after stimulation by CD40L. A, Anti-Ig induces nuclear B-binding activity in CD40L-stimulated xid B cells. B cells from xid and wt mice were cultured in medium (M) alone for 3 h (CD40L(-)/M), or were cultured in medium and then stimulated with anti-Ig at 15 µg/ml (Ig) or the combination of P/I at 100 and 600 ng/ml, respectively, for 3 h; B cells were cultured with CD40L/CD8 plus anti-CD8 for 48 h (CD40L(+)/M), or were cultured with CD40L and stimulated with Ig or P/I for the last 3 h of the 48-h culture period. Nuclei were isolated and extracted as described in Materials and Methods. Nuclear extract protein was tested for binding to a B 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 level of nuclear B-binding activity in anti-Ig- and P/I-treated samples over medium control values after normalization to NF-Y is provided below the corresponding lanes. Results are shown for one of three comparable experiments. B, CD40L and anti-Ig act sequentially to induce NF-B in xid B cells. xid B cells were cultured with CD40L/CD8 plus anti-CD8 for 48 h (M), or were cultured with CD40L and stimulated with Ig for the last 3 h of the 48-h culture period (Wash, -); xid B cells were also cultured with CD40L/CD8 plus anti-CD8 for 44 h, washed, incubated in medium for 1 h, and then stimulated with Ig (in the absence of CD40L) for 3 additional h (Wash, +). Nuclei were isolated and extracted as described in Materials and Methods. EMSA results are shown for one of two comparable experiments. C, The composition of NF-B induced by anti-Ig in CD40L-treated xid B cells is similar to that induced by anti-Ig in CD40L-treated control B cells. Nuclear extract protein samples were evaluated by EMSA. The identity of anti-Ig-induced B-binding nucleoprotein complexes 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.

The situation was very different for B cells previously treated with CD40L (Fig. 1A). Although B cell stimulation through CD40 induced nuclear NF-B early on as expected (24, 26, 27), the level returned to baseline within 2 days (Fig. 1 and data not shown). Addition of anti-Ig at this time strongly induced NF-B in xid B cells, to a level similar to that observed after anti-Ig stimulation of CD40L-treated control B cells. Comparable results were obtained when treatment with CD40L and stimulation by anti-Ig were separated by a wash and rest period consisting of incubation in medium without reagents for 1 (Fig. 1B) or more (data not shown) hours. The specificity of this Btk-independent, BCR-induced B-binding activity was confirmed by cold competition analysis in which unlabeled oligonucleotide containing a mutant B-binding site failed to effectively interfere with the formation of nucleoprotein complexes consisting of labeled wt B oligonucleotide and nuclear extract protein derived from either xid B cells or control B cells (data not shown). The nature of this BCR-induced B-binding activity was further explored through supershift analysis to identify participating Rel-related proteins (Fig. 1C). The principal NF-B components induced by anti-Ig stimulation of CD40L-treated xid B cells appeared to be p50 and c-Rel along with lesser amounts of immunoreactive p52, p65, and RelB. This same pattern was observed after anti-Ig stimulation of CD40L-treated control B cells (Fig. 1C) and was also evident in nuclear extracts obtained from anti-Ig-stimulated 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 B-binding nucleoprotein complexes. These results indicate that sIg engagement after CD40L treatment induces nuclear NF-B in xid B cells that is indistinguishable from NF-B induced by anti-Ig in control B cells, in direct contrast to the complete failure of anti-Ig to activate NF-B in untreated xid B cells.

BCR-mediated NF-B induction in CD40L-treated xid B cells is accompanied by IB degradation

To begin to characterize the mechanism responsible for Btk-independent NF-B activation in CD40L-treated B cells, we monitored levels of IB (28) before and after addition of anti-Ig. Results are shown in Fig. 2. Addition of anti-Ig to naive control B cells produced a time-dependent decline in IB, whereas sIg cross-linking in naive xid B cells produced little change in IB, as previously reported (8, 9). However, CD40L treatment for 24 (data not shown) or 48 h greatly altered the behavior of IB in response to sIg engagement, in that following CD40L, anti-Ig stimulation of xid B cells produced a decline in cellular IB that was as great as that observed with control B cells.

View larger version (32K): [in this window] [in a new window]   FIGURE 2. BCR-mediated NF-B induction in CD40L-stimulated xid B cells is accompanied by IB degradation. B cells from xid and wt control mice were cultured in medium alone for 1 h (CD40L(-)) or were cultured in medium and then stimulated with anti-Ig at 15 µg/ml (Ig) for 60 and 120 min; B cells were cultured with CD40L/CD8 plus anti-CD8 for 48 h (CD40L(+)), or were cultured with CD40L and stimulated with Ig. Whole cell extracts were prepared and Western blotted for IB. Blots were reprobed with anti-p38 to verify equal loading of extract protein. One of two comparable experiments is shown.

View larger version (76K): [in this window] [in a new window]   FIGURE 3. The level and composition of Rel-related proteins is similar in xid and wt control B cells treated with CD40L. B cells from xid and wt control mice were cultured in medium alone for 1 h or were cultured with CD40L/CD8 plus anti-CD8 (CD40L) for 48 h, as indicated. Whole cell extracts were prepared and Western blotted for Rel-related proteins as described in Materials and Methods. Blots were reprobed with anti-actin to verify equal loading of extract protein. One of two comparable experiments is shown.

To determine whether circumventing the block in BCR signaling for NF-B induction produced by mutant Btk extends to other aspects of B cell behavior, we evaluated proliferative responses. Previous reports indicate that xid B cells enter S phase poorly in response to sIg cross-linking (6, 7), but are stimulated by CD40 engagement (29, 30), so we evaluated responses to anti-Ig and CD40L alone and in combination. Results are shown in Fig. 4. B cells from xid mice incorporated [³H]thymidine in response to LPS and the combination of P/I much like B cells from control mice did. Although CD40L elicited less thymidine incorporation than LPS or P/I, the responses of xid and control B cells were again similar. However, xid B cells failed to respond to anti-Ig (as expected), whereas control B cells responded strongly. Remarkably, though, the combination of CD40L plus anti-Ig acted in synergy in xid B cells to produce substantial thymidine incorporation that was much greater than the sum of the responses to CD40L and to anti-Ig alone. This was true both when anti-Ig was present with CD40L during the last 24 h of a 48-h culture with CD40L (Fig. 4A), and when treatments with CD40L (24 h) and anti-Ig (24 h) were separated by a wash (Fig. 4B). These results indicate that CD40L treatment rescues not only defective NF-B induction, but also the poor proliferative response, of xid B cells to BCR cross-linking.

View larger version (30K): [in this window] [in a new window]   FIGURE 4. xid B cells proliferate in response to anti-Ig stimulation after treatment with CD40L. A, B cells from xid (left panel) and wt control (right panel) mice were cultured for 48 h with medium alone (M), anti-Ig at 15 µg/ml (Ig), PMA and ionomycin at 100 and 600 ng/ml, respectively (P/I), LPS at 25 µg/ml, and CD40L/CD8 plus anti-CD8 Ab (CD40L); the latter were treated with CD40L alone (CD40L/M) or with CD40L plus Ig or P/I added for the last 24 h of the 48-h culture period (CD40L/Ig and CD40L/P/I, respectively). Results represent the mean values of quadruplicate cultures; lines indicate SEMs. One of three comparable experiments is shown. B, xid B cells were cultured for 48 h with medium alone (M) or with anti-Ig at doses of 15, 5, and 1.5 µg/ml, as indicated (left-hand group). Separate B cell populations were cultured with CD40L for 24 h, washed, and then cultured with medium alone (M) or with anti-Ig at the indicated doses (CD40L (24 h); middle group). Additional B cell populations were cultured with CD40L alone for 48 h (CD40L/M), or with CD40L plus anti-Ig added for the last 24 h of the 48-h culture period at the indicated doses (CD40L (48 h); right-hand group), as in A. Incorporation of label was assessed after exposure of cells to [3H]thymidine during the last 6 h of culture. Results represent the mean values of quadruplicate cultures; lines indicate SEMs. One of three comparable experiments is shown.

	Discussion

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The mechanism by which BCR access to IB/NF-B in the absence of Btk is created by CD40 stimulation remains unknown. Similarly, little is known regarding the nature of the upstream events triggered by sIg cross-linking that circumvent the need for Btk. Notably, BCR-induced IB degradation occurred similarly in CD40L-stimulated xid and wt B cells, and the Rel-related protein composition of BCR-triggered nuclear B-binding activity (derived from comparable pools of NF-B) was similar in both groups as well. Thus, the Btk-independent BCR pathway merges with the classical Btk-dependent BCR pathway upstream of IB, but the level at which this occurs has not been clarified.

These results demonstrate that BCR-triggered signaling which is blocked by Btk mutation and consequent dysfunction can be reconstituted through the intervention of another surface receptor, CD40. This amounts to creation of a new, or marked enhancement of a previously underutilized or inaccessible, BCR signaling pathway. The general principle established here may be limited to the influence of CD40 on genetic blockade of one or more upstream elements of BCR-induced intracellular signaling, but raises the possibility that other loss-of-function signaling mutations might be normalized through stimulation of specific receptors.

	Footnotes

 
¹ This work was supported by U.S. Public Health Service Grants AI40181 and AI45112 awarded by the National Institutes of Health.

² Address correspondence and reprint requests to Dr. Thomas L. Rothstein, Immunobiology Unit, Evans Biomedical Research Center, Room 437, Boston University Medical Center, 650 Albany Street, Boston, MA 02118. E-mail address: trothstein{at}medicine.bu.edu

³ Abbreviations used in this paper: Btk, Bruton’s tyrosine kinase; xid, X-linked immunodeficiency; BCR, B cell receptor; sIg, surface Ig; P/I, PMA plus ionomycin; CD40L, CD40 ligand; wt, wild type.

Received for publication November 21, 2002. Accepted for publication January 29, 2003.

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