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The Journal of Immunology, 2003, 170: 3118-3124.
Copyright © 2003 by The American Association of Immunologists

NF-{kappa}B Is Required for Surface Ig-Induced Fas Resistance in B Cells1

Brian R. Schram*,{ddagger} and Thomas L. Rothstein2,*,{dagger},{ddagger}

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The susceptibility of primary murine B cells to Fas-mediated apoptosis is regulated in a receptor-specific fashion. Whereas CD40 engagement produces marked sensitivity to Fas killing, engagement of the B cell Ag receptor blocks Fas signaling for cell death in otherwise Fas-sensitive, CD40-stimulated targets and thus induces Fas resistance. The signaling pathway that leads from B cell Ag receptor to Fas resistance has not been fully characterized, but has been shown to depend on new gene expression. NF-{kappa}B is activated following B cell Ag receptor engagement and is associated with antiapoptosis; thus, it would seem a likely candidate to mediate transcriptional activation for inducible Fas resistance. Inhibition of B cell Ag receptor signaling for NF-{kappa}B activation completely blocked induction of Fas resistance by anti-Ig, and this same phenotype was observed both with chemical inhibitors such as lactacystin and pyrrolidinedithiocarbamate as well as with an I{kappa}B{alpha} dominant negative TAT fusion protein. Antiapoptotic, NF-{kappa}B-responsive transcripts include two gene products previously implicated in mediating anti-Ig-induced Fas resistance, Bcl-xL and FLIP. B cell Ag receptor-induced up-regulation of both these gene products was blocked by NF-{kappa}B inhibition, suggesting a mechanism by which the loss of nuclear NF-{kappa}B alters the sensitivity of B cell Ag receptor-stimulated B cells to Fas-mediated apoptosis. These results indicate that activation of NF-{kappa}B plays a key role in mediating Fas resistance produced by B cell Ag receptor engagement.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The Fas death receptor is a member of the TNF receptor family (1, 2, 3, 4). Interaction of the Fas receptor with Fas ligand (FasL)3 results in apoptotic death of the Fas-bearing cell. The consequences of defective Fas signaling are apparent in the lpr and gld mouse models (5, 6, 7, 8, 9, 10) and in the human disease autoimmune lymphoproliferative syndrome (11, 12, 13, 14, 15). These dyscrasias are characterized by high levels of serum Ig and autoantibody production, suggesting that Fas affects B cells directly. This is supported by work showing that the defect in B cell behavior associated with abnormal Fas expression is distinguishable from the influence of the lpr mutation on other immune cell populations (16, 17, 18). Separately, autoreactive B cells have been shown to be deleted by a Fas-dependent mechanism in adoptive transfer experiments (19). Taken together these results indicate that Fas-mediated apoptosis plays a key role in regulating B cell activity.

The relative susceptibility of B cells to Fas-mediated apoptosis is determined by the nature of signaling through additional receptors beyond Fas. In primary murine B cells, treatment with CD40 ligand (CD40L) or LPS results in increased sensitivity to Fas killing, due at least in part to up-regulation of surface Fas receptor expression (20, 21, 22, 23, 24, 25, 26). However, concurrent or subsequent treatment with anti-Ig Ab renders primary B cells relatively resistant to Fas-mediated apoptosis even in otherwise Fas-sensitive targets (24, 27, 28). Results with actinomycin D and cycloheximide indicate that new gene expression and new protein synthesis are required for induction of Fas resistance (29), and several gene products have been implicated in this process, including Bcl-xL, FLIP, and Fas apoptosis inhibitory molecule (30, 31, 32, 33, 34, 35). However, the signaling pathways leading from surface Ig (sIg) to induction of Fas resistance have not been clarified beyond the level of protein kinase C (29, 36).

The transcription factor NF-{kappa}B was originally identified as a protein that binds to an enhancer region of the Ig {kappa} light chain locus (37). NF-{kappa}B 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 (38). The latter two proteins are synthesized as larger precursors (of 105 and 100 kDa, respectively) that are proteolytically cleaved.

NF-{kappa}B plays a key role in cellular growth, differentiation, and stress responses aside from its role in regulating Ig gene expression (39). Although constitutively active in mature B cells, NF-{kappa}B is further induced following stimulation through sIg or CD40 (40, 41, 42, 43, 44). NF-{kappa}B activation is regulated by a family of inhibitor proteins (I{kappa}B) that normally retain NF-{kappa}B dimers within the cytosol in an inactive state (45). I{kappa}B{alpha} is the most well studied of the several inhibitor proteins. Following specific phosphorylation of N-terminal serines 32 and 36, I{kappa}B{alpha} becomes a target for ubiquitination and subsequent proteasomal degradation, thereby releasing NF-{kappa}B, which then localizes to the nucleus by virtue of a now exposed nuclear localization sequence. I{kappa}B{alpha} phosphorylation is mediated by the large IKK complex, which, in turn, may be phosphorylated by any of a number of kinases (39). Mutation of specific IKK-phosphorylated serines in I{kappa}B{alpha} yields an inhibitor protein that cannot be degraded and that acts as a dominant negative for NF-{kappa}B induction (46).

NF-{kappa}B mediates antiapoptotic signaling after TNF receptor engagement, and removal of NF-{kappa}B results in more efficient TNF-induced apoptosis (46, 47, 48). Further, deletion of RelA results in embryonic lethality due to extensive liver apoptosis, presumably due to TNF-{alpha} cytotoxicity (49, 50). Along the same lines, sIg-induced cell death in WEHI-231 B cells is opposed by signaling that induces or prolongs NF-{kappa}B activation (51). These results indicate that NF-{kappa}B produces an antiapoptotic effect, and this conclusion is supported by the identification of NF-{kappa}B-dependent genes whose protein products interfere with apoptotic cascades, such as Bcl-xL (52) and FLIP (53), both of which have been implicated in sIg-induced Fas resistance (30, 33). However, in some situations NF-{kappa}B induction appears to enhance apoptosis (54, 55), leading to uncertainty as to whether NF-{kappa}B acts to enhance or oppose Fas-mediated apoptosis in B cells. This uncertainty is compounded by the observation that NF-{kappa}B is strongly activated following stimulation through CD40 alone, which produces high level sensitivity to Fas killing. To clarify the role of NF-{kappa}B in influencing the outcome of Fas engagement in B cells, we used both chemical and biological inhibitors to block NF-{kappa}B activation. Here we report that NF-{kappa}B is required for sIg-induced resistance to Fas-mediated apoptosis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Male BALB/cByJ mice and male C3H/HeJ mice at 8–12 wk of age were obtained from The Jackson Laboratory (Bar Harbor, ME).

Isolation of B cells and stimulation

B cells were prepared from spleen cell suspensions by negative selection as described previously (40). Briefly, splenocytes were depleted of T cells by treatment with anti-Thy1.2 Ab, followed by complement lysis; the resulting cells were then subjected to Lympholyte-M (Cedarlane, Ontario, Canada) density separation to remove dead cells. B cells were cultured at 2–4 x 106 cells/ml in RPMI 1640 medium (BioWhittaker, Walkersville, MD), supplemented with 5% FBS (Sigma-Aldrich, St. Louis, MO), 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 cultured for 2 h and harvested for medium controls or were stimulated with F(ab')2 goat anti-mouse IgM and/or CD40L-CD8 fusion protein-containing supernatant (56), followed by cross-linking with anti-CD8 Ab. Optimal dilutions of the fusion protein and cross-linking Ab were determined experimentally in proliferation and cytotoxicity assays.

Preparation of nuclear extracts

Nuclear extracts were prepared from untreated and treated B cells as previously described (40, 42). Briefly, nuclei were collected after hypotonic lysis of B cells in a buffer containing 10 mM HEPES (pH 7.9), 1.5 mM MgCl2, and 0.5 mM DTT. Isolated nuclei were extracted with a high salt buffer (430 mM NaCl) for 45 min at 4°C. Nuclear debris was removed by centrifugation, and supernatant was aliquoted and stored at -80°C. All buffers contained a panel of protease inhibitors, including PMSF at 0.5 mM and leupeptin, aprotinin, antipain, chymostatin, and pepstatin A at 1 µg/ml. Protein concentrations were determined as described by Bradford using reagents obtained from Bio-Rad (Hercules, CA) (57).

EMSA

An NF-{kappa}B DNA binding element derived from the mouse Ig {kappa} light chain enhancer was prepared as previously described (40, 42) and labeled with [32P]dATP{alpha} by fill-in using the Klenow fragment of DNA polymerase I (Roche, Indianapolis, IN). Binding reactions were conducted at room temperature for 20 min as previously described (40, 42) and included 1 µg of nuclear extract plus the radiolabeled oligonucleotide. The resulting nucleoprotein complexes were separated from free probe by electrophoresis on 6% native polyacrylamide gels (30:1, acrylamide:bis-acrylamide) at 100 V. Gels were dried and visualized by autoradiography on X-OMAT-R film (Eastman Kodak, Rochester, NY).

Western immunoblot analysis

Whole-cell lysates were prepared from cultured primary B cells with RipA buffer (50 mM Tris (pH 7.4), 1% deoxycolate, 1% Triton X-100, 0.1% SDS, 1 mM EDTA (pH 8.0), and 150 mM NaCl). Protein concentrations were determined using the Bradford method; equivalent amounts of protein were electrophoresed on 12 or 15% SDS-polyacrylamide gels. Separated proteins were transferred to nitrocellulose membranes using a semidry transfer apparatus (Bio-Rad). Proteins were then detected with Abs specific for HA (Roche) and a HRP-linked secondary Ab (Amersham Pharmacia Biotech, Piscataway, NJ). The immunoreactive proteins were detected by ECL (Amersham Pharmacia Biotech) on X-OMAT-R film (Eastman Kodak).

Cell-mediated cytotoxicity

B cells (2 x 106) were cultured in 1.2 ml of supplemented RPMI 1640 in the wells of 24-well tissue culture plates (Corning, Acton, MA) for a total of 48 h at 37°C in 6% CO2. Susceptibility to Fas-mediated apoptosis was elicited by treatment with CD40L for the entire culture period as described. Resistance to Fas-mediated apoptosis was induced by adding F(ab')2 goat anti-mouse IgM (15 µg/ml) to CD40L-stimulated B cells 12 h prior to the end of the culture period; inhibitors were added 0.5 h before addition of anti-Ig (12.5 h prior to the end of the culture period). Stimulated B cells were then tested as targets for Fas-mediated apoptosis in standard lectin-dependent 4-h 51Cr release assays as previously described (24, 29), using AE7 CD4+ Th1 effector cells or soluble FasL.

Immunofluorescent staining

B cells were stained with Jo-2 anti-Fas Ab (BD PharMingen, San Diego, CA) and analyzed by flow cytometry on a FACScan instrument (BD Biosciences, Mountain View, CA) as previously described (58).

The pTAT construct and protein coupling

The plasmid containing I{kappa}B{alpha}DN was subjected to high fidelity PCR using Taq Gold (Promega, Madison, WI) to amplify the I{kappa}B{alpha} gene, and the resulting amplified sequence was cloned into the pTAT-HA bacterial expression vector (a kind gift from Dr. S. Dowdy, Washington University, St. Louis MO). The pTAT vector contains a six-histidine tag, an influenza hemagglutinin epitope tag followed by the TAT transduction domain, and finally the multiple cloning sequence (59). The resultant plasmid, pTAT-I{kappa}B{alpha}, was used to transform the DH5 strain of Escherichia coli. Transformants were screened for inserts initially by PCR and were subsequently sequenced. The pTAT-I{kappa}B{alpha}DN was then used to express the TAT-coupled I{kappa}B{alpha} fusion protein in the BL21 (DE3) strain of E. coli. Following 4–6 h of induction with 0.1 mM isopropyl {beta}-D-1-thiogalactospyranoside, the cells were sonicated in 8 M urea; the TAT-coupled I{kappa}B{alpha}DN was purified on a nickel-Sepharose column (Qiagen, Valencia, CA) and then applied to an ion exchange column (Mono Q) in 4 M urea. To shock-misfold the protein, the ion exchange column was switched in one step from 4 M urea to aqueous buffer (20 mM HEPES (pH 8.0)). TAT-I{kappa}B{alpha}DN was eluted with a gradient from 50 mM to 1 M NaCl, followed by desalting on a PD-10 column (Amersham Pharmacia Biotech, Sunnyvale, CA) into PBS and frozen in 10% glycerol at -80°C. The pTAT-green fluorescence protein (GFP) control plasmid used in these experiments was a kind gift from Dr. J. Tumang (Weill Medical College, Cornell University, New York, NY) and was purified in the same manner as the TAT-I{kappa}B{alpha}DN protein. The final protein concentration was estimated by SDS-PAGE in comparison with BSA standards stained with Coomassie Blue.

Reagents

F(ab')2 goat anti-mouse IgM was obtained from Jackson ImmunoResearch Laboratories (West Grove, PA), pyrrolidinedithiocarbamate (PDTC) was obtained from Sigma-Aldrich, and lactacystin (LC) was obtained from Biomol (Plymouth Meeting, PA). Soluble recombinant CD40L was obtained as supernatant from transfected J558L cells that secrete a chimeric CD40L/CD8{alpha} fusion protein (56) and was dialyzed as previously described (44). A similarly dialyzed supernatant containing anti-CD8 Ab from the 53-6-72 hybridoma was used to cross-link the fusion protein as previously described (44). Soluble FasL was obtained from ALEXIS Biochemicals (San Diego, CA). [32P]dATP{alpha} and [32P]dATP{gamma} were obtained from DuPont-NEN (Boston, MA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
NF-{kappa}B inhibitors LC and PDTC block sIg-mediated Fas resistance in B cells

To determine whether NF-{kappa}B plays a role in producing Fas resistance that results from sIg engagement, studies were conducted using the relatively specific inhibitors of NF-{kappa}B, LC (60, 61, 62) and PDTC (63). LC and PDTC are both toxic to B cells with prolonged incubation, and treatment results in cytotoxicity by 18 h (64). To operate within these limits, a period of 12 h was chosen for anti-Ig treatment. This period of sIg stimulation results in maximal or near-maximal Fas resistance, comparable to that obtained with longer periods of sIg engagement (29). LC or PDTC was added to B cells 30 min before the addition of anti-Ig, that is, 12.5 h before cells were harvested for cytotoxicity assay. Results with LC are shown in Fig. 1. B lymphocytes treated with CD40L for 48 h were quite sensitive to the effects of Fas engagement, and substantial cytotoxicity was evident. Notably, the addition of anti-IgM 12 h prior to the end of the culture period produced protection against Fas killing in these otherwise Fas-sensitive targets. The addition of LC in conjunction with anti-Ig largely reversed the Fas resistance afforded by sIg engagement, whereas the addition of LC to B cells stimulated by CD40L alone at the same time (12.5 h before the end of culture) had little effect on subsequent sensitivity to Fas killing. Thus, there was little difference in percent specific lysis after Fas engagement of B cells treated with CD40L alone, CD40L plus LC, or CD40L and anti-Ig plus LC. The loss of anti-Ig-induced Fas resistance produced by LC was not due to altered Fas expression, as this was elevated to a similar extent in all experimental conditions in which B cells were treated with CD40L (Fig. 1B). Lactacystin blocks NF-{kappa}B activation by disabling the proteasome, which is vital to many cellular processes aside from I{kappa}B degradation. Further, LC has been reported to activate caspase 8 (65). Thus, one potential explanation for the observed loss of Fas resistance is that LC produces cytotoxicity in addition to blocking NF-{kappa}B induction, indirectly obscuring the protective effect of anti-Ig. To further investigate the role of NF-{kappa}B a similar series of experiments was performed with the inhibitor PDTC, which blocks NF-{kappa}B activation in a manner distinct from LC and has no effect on other transcription factors (63). Results are shown in Fig. 2. The effect of PDTC on inducible Fas resistance was nearly identical to the results observed with LC, in that PDTC had little effect on the level of specific lysis produced by Fas engagement of B cells treated with CD40L alone, but eliminated the protection against Fas killing produced by anti-Ig. As with LC, PDTC did not alter the elevated level of Fas expression produced by CD40L. Thus, two inhibitors of NF-{kappa}B that operate through distinct mechanisms similarly inhibit the induction of Fas resistance produced by sIg engagement.



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  		FIGURE 1. NF-{kappa}B inhibitor lactacystin blocks sIg-induced Fas resistance in B cells. A, Primary B cells from BALB/cByJ mice were cultured with CD40L/CD8{alpha} fusion protein cross-linked with anti-CD8 Ab for 48 h (CD40L) or were cultured with CD40L for 48 h plus F(ab')2 goat anti-mouse IgM Ab at 10 µg/ml (anti-Ig), present during the last 12 h of culture, with or without LC at 10 µM added 0.5 h before anti-Ig, as indicated. B cells were then radiolabeled and tested as targets for Fas-induced cytotoxicity mediated by AE7 CD4+ Th1 effector cells in standard lectin-dependent 51Cr release assays at various E:T cell ratios, as indicated. Results are shown for one of five comparable experiments. For each condition the mean percentage of specific cell lysis of triplicate assays is displayed along with a line indicating the SD. Spontaneous 51Cr release values were: CD40L, 21%; CD40L with LC, 17%; CD40L plus anti-Ig, 18%; and CD40L plus anti-Ig with LC, 21%. B, B cells treated as described above were stained with FITC-labeled anti-Fas Ab ({blacksquare}) or an irrelevant control Ab ({square}), and fluorescence intensity was assessed by flow cytometry.

 


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  		FIGURE 2. NF-{kappa}B inhibitor PDTC blocks sIg-induced Fas resistance in B cells. A, Primary B cells from BALB/cByJ mice were cultured with CD40L/CD8{alpha} fusion protein cross-linked with anti-CD8 Ab for 48 h (CD40L) or were cultured with CD40L for 48 h plus F(ab')2 goat anti-mouse IgM Ab at 10 µg/ml (anti-Ig) present during the last 12 h of culture, with or without PDTC at 5 mM, added 0.5 h before anti-Ig, as indicated. B cells were then radiolabeled and tested as targets for Fas-induced cytotoxicity mediated by AE7 CD4+ Th1 effector cells in standard lectin-dependent 51Cr release assays at various E:T cell ratios as indicated. Results are shown for one of five comparable experiments. For each condition the mean percentage of specific cell lysis of triplicate assays is displayed along with a line indicating the SD. Spontaneous 51Cr release values were: CD40L, 18%; CD40L with PDTC, 27%; CD40L plus anti-Ig, 19%; and CD40L plus anti-Ig with PDTC, 25%. B, B cells treated as described above were stained with FITC-labeled anti-Fas Ab ({blacksquare}) or an irrelevant control Ab ({square}), and fluorescence intensity was assessed by flow cytometry.

 
TAT-I{kappa}B{alpha}DN fusion protein blocks NF-{kappa}B induction in primary B lymphocytes

To more selectively inhibit NF-{kappa}B activation while limiting interaction with other metabolic pathways, a TAT-I{kappa}B{alpha}DN fusion protein was constructed for use in experiments similar to those described above. The fusion protein incorporates a short peptide sequence from the HIV TAT protein, which allows uptake of proteins of a certain size into virtually all cells (59). To demonstrate the capacity of TAT-I{kappa}B{alpha}DN to block NF-{kappa}B activation, nuclear extracts were prepared from B cells that had been treated with the fusion protein and then stimulated with anti-Ig. To model the regimen used for induction of Fas resistance, B cells were cultured with CD40L for 48 h and were stimulated with anti-Ig for the last 3 h of culture, the latter because NF-{kappa}B induction produced by anti-Ig is maximal after this time period. Results are shown in Fig. 3. CD40L treatment induced nuclear NF-{kappa}B, the level of which at 48 h was still higher than that present in unmanipulated B cells (data not shown). Stimulation of CD40L-treated B cells with anti-Ig then produced a further increase in NF-{kappa}B, which in separate experiments amounted to 2-fold, as determined by Western blotting nuclei for c-Rel content (data not shown). However, the addition of TAT-I{kappa}B{alpha}DN completely eliminated the increase in NF-{kappa}B produced by anti-Ig. The outcome of TAT-I{kappa}B{alpha}DN treatment was similar to the inhibition of NF-{kappa}B induction observed after treatment with PDTC. Inhibition by TAT-I{kappa}B{alpha}DN and that by PDTC were specific, inasmuch as neither inhibitor affected transcription factor binding to a consensus AP-1 site (data not shown).



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  		FIGURE 3. TAT-I{kappa}B{alpha}DN blocks sIg-mediated induction of nuclear NF-{kappa}B. Primary B cells were cultured with CD40L/CD8{alpha} fusion protein cross-linked with anti-CD8 Ab for 48 h (CD40L) or were cultured with CD40L for 48 h plus F(ab')2 goat anti-mouse IgM Ab at 10 µg/ml (anti-Ig) present during the last 2 h of culture, with or without TAT-I{kappa}B{alpha}DN fusion protein added 0.5 h before anti-Ig. Nuclear extracts were prepared from these samples and examined for transcription factor expression by EMSAs using radiolabeled oligonucleotides containing consensus sequences for NF-{kappa}B binding, as indicated by arrows. Results are shown for one of three comparable experiments.

 
Loss of NF-{kappa}B activity correlates with loss of inducible Fas resistance

TAT-I{kappa}B{alpha}DN was then used in a series of experiments to determine the effect of blocking NF-{kappa}B activation on inducible Fas resistance. The fusion protein was added 30 min before the induction of Fas resistance by the addition of anti-Ig, in a protocol identical to that used for LC and PDTC. Results are shown in Fig. 4. As previously observed in Figs. 1 and 2, B cells cultured with CD40L alone were very susceptible to Fas-induced cytotoxicity, whereas B cells treated with both CD40L and anti-Ig were relatively resistant to Fas killing. This anti-Ig-induced Fas resistance was completely abrogated by TAT-I{kappa}B{alpha}DN. Notably, TAT-I{kappa}B{alpha}DN had little or no effect on the susceptibility to Fas killing of B cells cultured with CD40L alone. Further, the degree to which TAT-I{kappa}B{alpha}DN reversed anti-Ig-induced Fas resistance was similar to that produced by PDTC and was not reproduced by control TAT-GFP, which had no effect.



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  		FIGURE 4. TAT-I{kappa}B{alpha}DN blocks sIg-induced Fas-resistance in B cells. A, Primary B cells from BALB/cByJ mice were cultured with CD40L/CD8{alpha} fusion protein cross-linked with anti-CD8 Ab for 48 h (CD40L) or were cultured with CD40L for 48 h plus F(ab')2 goat anti-mouse IgM Ab at 10 µg/ml (anti-Ig), which was present during the last 12 h of culture, with or without TAT-I{kappa}B{alpha}DN fusion protein (3 ng/ml), TAT-GFP (3 ng/ml), or PDTC (5 mM), added 0.5 h before anti-Ig, as indicated. B cells were then radiolabeled and tested as targets for Fas-induced cytotoxicity mediated by soluble FasL at the doses indicated in standard 51Cr release assays. Results are shown for one of five comparable experiments. For each condition the mean percentage of specific cell lysis of triplicate assays is shown along with a line indicating the SD. B, Primary B cells from C3H/HeJ mice were cultured as described above with CD40L, anti-Ig, TAT-I{kappa}B{alpha}DN, and TAT-GFP, as indicated. B cells were then radiolabeled and tested as targets for Fas-dependent cytotoxicity mediated by soluble FasL at the doses indicated in standard 51Cr release assays. Results are shown for one of three comparable experiments. For each condition the mean percentage of specific cell lysis of triplicate assays is shown along with a line indicating the SD. Spontaneous 51Cr release values were: CD40L, 10%; CD40L with TAT-I{kappa}B{alpha}DN, 11%; CD40L plus anti-Ig, 13%; and CD40L plus anti-Ig with TAT-I{kappa}B{alpha}DN, 14%.

 
To further ensure that inhibition of inducible Fas resistance was not due to contamination of purified TAT-I{kappa}B{alpha}DN by bacterial products, experiments similar to those described above were conducted using C3H/HeJ mice that are defective in Toll-like receptor 4 signaling and are unresponsive to LPS (66, 67, 68). Results are shown in Fig. 4B. TAT-I{kappa}B{alpha}DN reversed sIg-induced Fas resistance in C3H/HeJ B cells. This results along with the failure of TAT-GFP to affect the induction of Fas resistances strongly suggests that bacterial products do not play a role in antagonizing the induction of Fas resistance, although this possibility is not entirely ruled out because C3H/HeJ B cells bear other Toll-like receptors.

B cell treatment with TAT-I{kappa}B{alpha}DN reduces the expression of antiapoptotic molecules

To elucidate the mechanism by which TAT-I{kappa}B{alpha}DN blocks the induction of Fas resistance, the expression of several key antiapoptotic molecules was evaluated. B cells were treated with fusion protein 30 min before the addition of anti-Ig; whole-cell lysates were then prepared 12 h later and probed for various molecules by Western blotting. Results are shown in Fig. 5. Bcl-xL expression was strongly induced when anti-Ig was added to CD40L, and this induction was strongly inhibited by the fusion protein. Similarly, c-FLIP expression was induced when anti-Ig was added to CD40L-stimulated B cells, and this induction was dramatically reduced by TAT-I{kappa}B{alpha}DN. In contrast, Bcl-2 expression was little affected by TAT-I{kappa}B{alpha}DN (data not shown). These results suggest that Bcl-xL and c-FLIP contribute, in whole or in part, to the Fas resistance generated by anti-Ig in a {kappa}B-dependent fashion.



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  		FIGURE 5. TAT-I{kappa}B{alpha}DN blocks sIg-mediated induction of Bcl-xL and c-FLIP. Primary B cells from BALB/cByJ mice were cultured with CD40L/CD8{alpha} fusion protein cross-linked with anti-CD8 Ab for 48 h (CD40L) or were cultured with CD40L for 48 h plus F(ab')2 goat anti-mouse IgM Ab at 10 µg/ml (anti-Ig), present during the last 12 h of culture, with or without TAT-I{kappa}B{alpha}DN fusion protein at 2.5 ng/ml or PDTC at 5 mM added 0.5 h before anti-Igs as indicated. In addition, B cells were cultured in medium alone for 2 h. Whole-cell extracts were prepared from each sample, and proteins were size-separated by SDS-PAGE and transferred to nitrocellulose. Membranes were blotted with either anti-Bcl-xL (A) or anti-FLIP Abs (B). Ab binding was detected with HRP-conjugated secondary Ab detected by chemiluminescence. The same blots were reprobed with actin-specific Abs to determine loading variation. The experiment displayed in A included B cells cultured with CD40L for 48 h plus anti-Ig present during the last 24 h of culture, indicated as CD40L + anti-Ig (24 ). Results are shown for one of three comparable experiments each for Bcl-xL and FLIP.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The work presented in this study demonstrates that interruption of sIg signaling for NF-{kappa}B activation is accompanied by interruption of sIg signaling for Fas resistance in primary B cells. This same phenotype was observed when NF-{kappa}B was blocked by both chemical and biological inhibitors, and the use of three different agents to interrupt NF-{kappa}B induction increases confidence in these results. The conclusion that NF-{kappa}B induction is required for sIg-mediated Fas resistance is strengthened by the previously described role of NF-{kappa}B in opposing TNF-mediated apoptosis and the reported transcriptional activation of several key antiapoptotic genes by NF-{kappa}B. Antiapoptotic, NF-{kappa}B-responsive transcripts include two gene products that were previously implicated in mediating anti-Ig-induced Fas resistance, Bcl-xL and FLIP (30, 33). Further, sIg-induced up-regulation of both these gene products was blocked by NF-{kappa}B inhibition, suggesting a mechanism by which the loss of nuclear NF-{kappa}B altered the sensitivity of B cells to Fas-mediated apoptosis. This does not, however, exclude a role for other antiapoptotic, NF-{kappa}B-responsive genes or other antiapoptotic genes whose NF-{kappa}B dependence is unknown, such as Fas apoptosis inhibitory molecule (34, 35), which were not tested in this work.

The role of NF-{kappa}B in mediating Fas resistance was previously tested in a different way by Owyang et al. (69), who examined the influence of c-Rel deletion on Fas sensitivity. The loss of c-Rel had little effect on the degree to which anti-Ig diminished the susceptibility to Fas-mediated apoptosis of anti-CD40-stimulated primary B cells despite the associated loss of Bcl-xL expression. At the same time, anti-Ig-induced up-regulation of FLIP expression was not affected by c-Rel deficiency. This work suggests that Bcl-xL either does not play a substantial role in sIg-induced Fas resistance or is fully compensated by FLIP and/or other sIg-induced antiapoptotic molecules. However, in the face of constitutive absence of c-Rel, enhanced expression of other antiapoptotic genes may contribute to the lack of a requirement for Bcl-xL in mediating sIg-induced Fas resistance in this instance. Alternatively, the loss of only c-Rel may differ from the loss of all {kappa}B-binding species in terms of the variety of {kappa}B-inducible apoptotic regulatory genes affected, as exemplified by FLIP.

As with primary B cells, up-regulation of FLIP expression appears to play a dominant role in mediating Fas resistance produced by anti-Ig in the A20 B cell line (33). Although it was suggested at one point that Fas resistance in A20 cells results from a failure of Fas-associated death domain protein association with Fas (70), induction of Fas resistance was not blocked by cycloheximide in that study, in marked contrast to the situation with primary B cells (29), and subsequent work with A20 cells demonstrated that Fas-associated death domain protein associates normally with Fas even after anti-Ig treatment (33). Most importantly, the reported role of FLIP in A20 cells as well as in primary B cells is entirely consistent with our results on the need for NF-{kappa}B in establishing Fas resistance, inasmuch as FLIP induction is NF-{kappa}B dependent (53), and thus supports the idea that NF-{kappa}B-mediated transcriptional activation is largely responsible for physiological Ag receptor-induced resistance to Fas-mediated apoptosis.

Previously, we and others found that CD40 engagement induces NF-{kappa}B activation. Inasmuch as NF-{kappa}B is here shown to play a key role in mediating B cell Ag receptor-driven down-regulation of Fas sensitivity, the question may be posed as to why the susceptibility to Fas killing of B cells stimulated through CD40 is not similarly modulated by CD40-induced NF-{kappa}B. Subtle differences exist in the Rel-related protein composition of {kappa}B-binding activity induced by engagement of sIg as opposed to CD40 (71, 72). Thus, a potential explanation for CD40-induced Fas sensitivity in the face of CD40-induced NF-{kappa}B is that the latter differs from sIg-induced NF-{kappa}B sufficiently (in terms of composition, level, or duration) to yield a nonidentical effect on the stimulated expression of key antiapoptotic molecules. Along these lines, B cell FLIP is not up-regulated by CD40 engagement, whereas it is substantially up-regulated by sIg cross-linking (33). Another potential explanation is that induction of certain antiapoptotic molecules may depend on B cell Ag receptor-specific transcription factors other than NF-{kappa}B, alone or in combination with NF-{kappa}B. Alternatively, the antiapoptotic effect of CD40-induced NF-{kappa}B may simply be overwhelmed by CD40-triggered proapoptotic influences. In this model the level of susceptibility to Fas killing would reflect the relative balance between pro- and antiapoptotic molecules that exists before Fas engagement. Thus, CD40-induced NF-{kappa}B may produce a level of antiapoptosis similar to that produced by sIg-induced NF-{kappa}B that remains inapparent because of the overall proapoptotic tendency of CD40-activated B cells, while at the same time the observable antiapoptotic effect of sIg engagement requires NF-{kappa}B. Unfortunately each of the NF-{kappa}B inhibitors used in this study, LC, PDTC, and TAT-I{kappa}B{alpha}DN, proved toxic when applied to B cells for >18 h (perhaps because the antiapoptotic effect of NF-{kappa}B was opposed), which precluded evaluation of NF-{kappa}B-inhibited B cells stimulated by CD40L, to induce Fas sensitivity. Although the present work has demonstrated the key role of NF-{kappa}B in mediating resistance to Fas killing produced by sIg engagement, the contribution of CD40-stimulated NF-{kappa}B to the regulation of primary B cell Fas sensitivity remains unclear at this time.


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

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

3 Abbreviations used in this paper: FasL, Fas ligand; CD40L, CD40 ligand; GFP, green fluorescence protein; LC, lactacystin; PDTC, pyrrolidinedithiocarbamate; sIg, surface Ig. Back

Received for publication July 23, 2002. Accepted for publication January 13, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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