The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tumang, J. R.
Right arrow Articles by Rothstein, T. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tumang, J. R.
Right arrow Articles by Rothstein, T. L.
The Journal of Immunology, 2002, 168: 2712-2719.
Copyright © 2002 by The American Association of Immunologists

BCR Engagement Induces Fas Resistance in Primary B Cells in the Absence of Functional Bruton’s Tyrosine Kinase1

Joseph R. Tumang2,*,{ddagger}, Robert S. Negm2,3,*,{ddagger}, Laura A. Solt{ddagger}, Thomas J. Schneider4,{dagger},{ddagger}, Thomas P. Colarusso{ddagger}, William D. Hastings{dagger},{ddagger}, Robert T. Woodland§ and Thomas L. Rothstein5,*,{dagger},{ddagger}

Departments of * Medicine and {dagger} Microbiology, Boston University School of Medicine, and {ddagger} Immunobiology Unit, Evans Memorial Department of Clinical Research, Boston University Medical Center, Boston, MA 02118; and § Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, MA 01655


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
B cell susceptibility to Fas-mediated apoptosis is regulated in a receptor-specific fashion. CD40 engagement produces marked sensitivity to Fas killing, whereas surface Ig (sIg) engagement blocks Fas signaling for cell death in otherwise sensitive, CD40-stimulated B cell targets, and thus, induces a state of Fas resistance. The signaling mediator, Bruton’s tyrosine kinase (Btk), is required for certain sIg-triggered responses, and Btk is reported to directly bind Fas and block Fas-mediated apoptosis. For these reasons, the role of Btk as a mediator of sIg-induced Fas resistance was examined. Dysfunction of Btk through mutation, and absence of Btk through deletion did not interfere with induction of Fas resistance by anti-Ig. This may be due, at least in part, to induction of Btk-dependent Bcl-2 family members by anti-Ig after CD40 ligand treatment. However, the susceptibility to Fas-mediated apoptosis of B cell targets stimulated by CD40 ligand alone was increased in the absence of Btk. These results indicate that Fas resistance produced by sIg triggering does not require Btk, but suggests that in certain situations Btk modulates B cell susceptibility to Fas killing.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The Fas (CD95) death receptor appears to play a principal role in immune homeostasis, inasmuch as mutation or deletion of Fas is associated with autoimmunity and progressive lymphadenopathy, both in mouse and in human (1, 2, 3). Fas deficiency produces dysfunction specifically within the B cell pool that is independent of the influence of Fas on other immune cells (4, 5, 6, 7), and may relate to the role of Fas triggering in deleting autoreactive B cells (8). Both in vitro and in vivo studies indicate that the susceptibility of B cell targets to Fas killing is regulated in a receptor-specific fashion (9, 10, 11, 12, 13, 14, 15). In particular, CD40 signaling produces up-regulation of Fas expression and increased sensitivity to Fas-induced cell death, whereas signaling through surface Ig (sIg)6 suppresses Fas-mediated apoptosis, even in otherwise sensitive CD40-stimulated B cells, and thus induces a state of Fas resistance. Fas resistance likely ensures the viability of B cells during critical, early interactions with activated, Fas ligand (FasL)-expressing T cells. In keeping with this, Fas resistant B cells are better APCs than Fas-sensitive B cells (16). Furthermore, Fas-resistant B cells block activation-induced cell death within the T cell pool (17). These latter results suggest that Fas resistance may not only enhance B cell responses, but may promote and prolong T cell responses as well. Notably, the threshold of sIg cross-linking required for induction of Fas resistance is higher for autoreactive B cells than for foreign Ag-specific B cells (18), which may play a role in preventing serological autoimmunity under normal circumstances, although aberrant acquisition of Fas resistance might represent an etiologic factor in the development of autoimmune dyscrasias.

Because of the presumed involvement of Fas resistance in normal and autoimmune responses, studies have been directed toward elucidating the mechanism by which sIg signaling modulates the sensitivity of B cells to Fas-mediated apoptosis. This work indicates that sIg-induced Fas resistance depends on activation of protein kinase C and on new macromolecular synthesis (13). Terminal effectors of B cell Fas resistance reportedly include Bcl-xL, c-FLIP, and the recently described novel anti-apoptotic gene product, Fas apoptosis inhibitory molecule (Refs. 19, 20, 21, 22 ; our unpublished observations). Expression of each of these gene products is up-regulated coordinately with induction of Fas resistance, and each is capable of blocking Fas-mediated apoptosis when overexpressed in isolation in primary B cells or B cell lines. Other, as yet unidentified, molecules may be involved as well.

The Tec kinase Bruton’s tyrosine kinase (Btk) is expressed in all hematopoietic lineages except T cells and represents a key intermediary for sIg-derived signaling (23, 24, 25). Mutation of Btk results in severely blocked B cell development in humans afflicted by X-linked agammaglobulinemia, but a milder form of B cell deficiency in mice with X-linked immunodeficiency (xid) (26, 27, 28, 29). Btk is a substrate for Lyn kinase as well as other Src family kinases, and its activity is influenced by pleckstrin homology domain-mediated binding to the phosphoinositide 3-kinase product phosphatidylinositol-3,4,5-trisphosphate, such that disruption of the p85{alpha} subunit results in a B cell immunodeficiency remarkably similar to the phenotype of xid and Btk-deficient mice (30, 31, 32, 33, 34, 35, 36). Btk in turn is a principal activator of phospholipase C-{gamma}2, and sIg-triggered phosphoinositide hydrolysis and Ca2+ responses are markedly diminished in xid B cells (37, 38, 39). The central role that Btk plays in directing downstream effects produced by sIg triggering is demonstrated by the complete failure of proliferation following anti-Ig treatment of B cells obtained from xid and Btk-/- mice (40, 41). Thus, it might be thought that sIg signaling for Fas resistance, like other outcomes of sIg engagement, depends on Btk.

Recently, it was reported that Btk modulates the sensitivity of B cells to Fas-mediated apoptosis (42). In chicken DT40 B cells, Btk bound Fas directly, interfering with signaling for cell death, such that Btk-deficient DT40 cells were much more sensitive to Fas killing than wild-type (WT) control cells. Combined with the observation that Btk protein expression was up-regulated following sIg engagement (43), these results raise the possibility that Btk may be responsible for sIg-induced Fas resistance, either as a signaling intermediary, or as a terminal inhibitor of the Fas death pathway. The present study was conducted to examine the role of Btk in promoting sIg-induced Fas resistance, and the capacity of Btk to modulate the sensitivity of B cells to Fas killing.


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

Male (CBA/N x A.By)F1 xid mice were bred and maintained at the University of Massachusetts Medical School (Worcester, MA). Male Btk-deficient and control C57BL/6, and additional xid and control CBA mice, were obtained at 8–10 wk of age from The Jackson Laboratory (Bar Harbor, ME).

B cell purification

B cells were prepared from spleen cell suspensions by negative selection, as previously described (9). Purified B cells were cultured at 37°C with 5% CO2 in RPMI 1640 medium (BioWhittaker, Walkersville, MD) supplemented with 5% heat-inactivated FBS (Sigma Aldrich, St. Louis, MO), 10 mM HEPES (pH 7.2), 50 µM 2-ME, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin.

Cell-mediated cytotoxicity

Following stimulation, B cells were tested as targets for Fas-dependent cytotoxicity in standard lectin-dependent 4 h 51Cr release assays using V{beta} 2 or AE7 CD4+ Th1 effector cells, as previously described (9). 51Cr release assays were also conducted using soluble FasL (sFasL) to induce cytotoxicity over a 4- to 8-h period.

Flow cytometric analysis

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 (12).

RNase protection assay

RNA was obtained from stimulated and unstimulated B cells using Ultraspec reagent (Biotecx Laboratories, Houston, TX) and DNase treated. The expression of Bcl-2 family members was assessed with 2.5 µg total RNA, using the mAPO-2 multitemplate probe set (BD PharMingen), as described by the manufacturer.

Reagents

Soluble recombinant CD40 ligand (CD40L) was obtained from transfected J558L cells that secrete a chimeric CD40L/CD8{alpha} fusion protein (44), as previously described (45). A similarly dialyzed supernatant containing anti-CD8 Ab from the 53-6-72 hybridoma was used to cross-link the fusion protein, as described (45). Affinity-purified F(ab')2 of polyclonal goat anti-mouse IgM (anti-Ig) were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). PMA and ionomycin were obtained from Sigma Aldrich. sFasL was obtained from ALEXIS Biochemicals (San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The role of Btk in mediating sIg-induced Fas resistance was evaluated by testing the capacity of anti-Ig treatment to block Fas-mediated apoptosis in CD40L-stimulated B cells containing either mutant (xid) or deleted (Btk knockout (KO)) forms of Btk. The combination of a protein kinase C-activating phorbol ester, PMA, and a calcium ionophore, ionomycin, which are together mitogenic for mutant Btk and Btk-deficient B cells (41, 46), was used to produce Fas resistance by triggering signaling intermediates downstream of Btk. As part of this study, the role of Btk in modulating B cell sensitivity to Fas killing after stimulation by CD40L alone was defined.

Fas-mediated apoptosis in xid B cells

B cells from xid and littermate control mice were stimulated with CD40L/CD8{alpha} cross-linked with anti-CD8 Ab (CD40L) for a total of 48 h, with or without anti-Ig or the combination of PMA plus ionomycin (P/I) added during the last 24 h of culture. B cells were then tested for Fas sensitivity by lectin-dependent chromium release assays in which cytotoxicity was produced by FasL-bearing CD4+ Th1 effector cells that kill in a Fas-dependent fashion (9). The results of two such experiments are displayed in Fig. 1Go. Stimulation with CD40L alone produced marked sensitivity to Th1 cell-mediated cytotoxicity in both WT control and in xid B cells. This is consistent with previous results indicating that some aspects of CD40 signaling remain intact in Btk mutant B cells (47, 48). As expected, the addition of anti-Ig to CD40L-stimulated control B cells led to a marked diminution of subsequent Fas-dependent cytotoxicity; that is, anti-Ig produced Fas resistance. Surprisingly, the same was true of xid B cells. Anti-Ig produced similar levels of Fas resistance in xid and in control B cells. The combination of P/I also produced Fas resistance in both control and xid B cells, and there was no consistent difference in the relative effectiveness of anti-Ig and P/I in producing Fas resistance in xid as compared with control B cells.



View larger version (34K):
[in this window]
[in a new window]
  		FIGURE 1. sIg signaling induces Fas resistance in xid B cells. Primary splenic B cells obtained from xid (Xid) or WT control (WT) 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 either F(ab')2 fragments of goat anti-mouse IgM at 10 µg/ml (CD40L/{alpha}Ig), or the combination of PMA at 100 ng/ml and ionomycin at 600 ng/ml (CD40L/P + I), added during the last 24 h of culture, as indicated. B cells were then radiolabeled and tested as targets for Fas-dependent cytotoxicity mediated by V{beta}2 CD4+ Th1 effector cells in standard lectin-dependent 51Cr release assays. Results obtained at E:T cell ratios of 3:1 and 1:1 are shown for two separate experiments. For each condition, the mean percentage of specific cell lysis of triplicate assays is shown, along with a line indicating the SEM.

 
To confirm that resistance to Th1 cell-mediated cytotoxicity induced by anti-Ig in xid B cells resulted from blocking Fas signaling for cell death, additional chromium release assays were conducted in which apoptosis was produced by triggering Fas with sFasL. The mean results of three such experiments, shown in Fig. 2Go, essentially recapitulate the results described above. Stimulation with CD40L alone produced marked sensitivity to FasL-induced cytotoxicity, and anti-Ig treatment produced protection against FasL-induced cytotoxicity, in both control and xid B cells. The level of Fas resistance produced by anti-Ig (that is, the magnitude of the reduction in Fas-mediated apoptosis derived by contrasting B cells treated with CD40L plus anti-Ig and B cells treated with CD40L alone) was not different for xid as compared with control B cells. One experiment from this set is shown in more detail in Fig. 3Go, which includes the results of FasL dose titration. At each dose of FasL, anti-Ig treatment reduced the level of Fas killing both for control and for xid B cells. As previously reported (9), B cell Fas expression was up-regulated by treatment with CD40L, but little changed by the addition of anti-Ig or by the addition of P/I after CD40L stimulation (Fig. 3GoB); this was true both of normal control and xid B cells, and thus, induction of Fas resistance in the presence of mutant Btk cannot be explained by shifts in Fas expression.



View larger version (25K):
[in this window]
[in a new window]
  		FIGURE 2. sIg signaling induces resistance to sFasL in xid B cells. Primary splenic B cells obtained from xid (Xid) or WT control (WT) 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 either F(ab')2 fragments of goat anti-mouse IgM at 10 µg/ml (CD40L/{alpha}Ig), or the combination of PMA at 100 ng/ml and ionomycin at 600 ng/ml (CD40L/P + I), added during the last 24 h of culture, as indicated. B cells were then radiolabeled and tested as targets for Fas-dependent cytotoxicity mediated by sFasL (50 ng/ml) in standard 51Cr release assays. The means of specific lysis for three independent experiments are shown, along with lines indicating the SEM.

 


View larger version (40K):
[in this window]
[in a new window]
  		FIGURE 3. sIg signaling induces resistance in xid B cells at various doses of sFasL. Primary splenic B cells obtained from xid (Xid) or WT control (WT) mice were treated as described in the legend to Fig. 2Go. Separate aliquots of B cells were radiolabeled and tested as targets for susceptibility to Fas-dependent cytotoxicity at the doses of sFasL indicated, and were stained with FITC-labeled Jo-2 monoclonal anti-Fas Ab and analyzed for Fas expression by flow cytometry. A, Mean percentage of specific cell lysis of triplicate assays for each condition, along with a line indicating the SEM. B, Relative cell number as a function of fluorescence intensity for each condition. The results of a single representative experiment are shown.

 
Fas-mediated apoptosis in Btk-deficient B cells

B cells from Btk-deficient and control mice were stimulated with CD40L for 48 h, with or without anti-Ig or the combination of P/I added during the last 24 h of culture, and then tested for Fas sensitivity with CD4+ Th1 effector cells, as in the experiments described above. The mean results of four experiments are displayed in Fig. 4Go. Stimulation with CD40L alone produced sensitivity to Fas-dependent cytotoxicity similarly in both control and in Btk-deficient B cells. However, particularly at higher E:T cell ratios, Btk-deficient B cells appeared to be somewhat more susceptible to Fas-mediated apoptosis than control B cells, after stimulation with CD40L alone. Regardless, anti-Ig addition produced equivalent levels of resistance to Fas killing in Btk-deficient and control B cells. This is especially apparent in comparing E:T cell ratios that produced similar levels of Fas sensitivity in B cells stimulated by CD40L alone, without the addition of anti-Ig. In other words, in comparing Btk-deficient B cells tested at an E:T cell ratio of 1:1 with control B cells tested at an E:T cell ratio of 3:1 (Fig. 4Go). The combination of P/I also produced Fas resistance in both normal and Btk-deficient B cells. These results indicate that, as with xid B cells, the induction of Fas resistance by sIg engagement is not abrogated by the loss of Btk function.



View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 4. sIg signaling induces Fas resistance in Btk-deficient B cells. Primary splenic B cells obtained from Btk-deficient (Btk KO) or WT control (WT) mice were cultured with CD40L/CD8{alpha} fusion protein cross-linked with anti-CD8 Ab for 48 h (CD40L), or with CD40L for 48 h plus either F(ab')2 fragments of goat anti-mouse IgM at 10 µg/ml (CD40L/{alpha}Ig ), or the combination of PMA at 100 ng/ml and ionomycin at 600 ng/ml (CD40L/P + I), added during the last 24 h of culture, as indicated. B cells were then radiolabeled and tested as targets for Fas-dependent cytotoxicity mediated by CD4+ Th1 effector cells in standard lectin-dependent 51Cr release assays. Results obtained at E:T cell ratios of 3:1 and 1:1 are shown. For each condition, the means of specific lysis for four independent experiments are shown, along with lines indicating the SEM.

 
To confirm that resistance to Th1 cell-mediated cytotoxicity induced by anti-Ig in Btk-deficient B cells resulted from blocking Fas signaling for cell death, additional chromium release assays were conducted in which apoptosis was produced by triggering Fas with sFasL. The mean results of three such experiments, shown in Fig. 5Go, again demonstrate induction of resistance to Fas-mediated apoptosis by anti-Ig in the absence of Btk. Although Btk-deficient B cells stimulated by CD40L alone were more sensitive to Fas-mediated apoptosis than were CD40L-stimulated control B cells (at a dose of 50 ng/ml sFasL), the reduction in Fas sensitivity produced by anti-Ig treatment was similar in comparing Btk-deficient with control B cells. One experiment from this set is shown in more detail in Fig. 6Go, which includes the results of FasL dose titration. As observed with the pooled results, Btk-deficient B cells were more sensitive than control B cells to Fas-induced cytotoxicity at each dose of FasL tested. Still, at each dose, anti-Ig treatment reduced the level of Fas killing for Btk-deficient as well as for control B cells. As noted above, B cell Fas expression was markedly up-regulated in xid and in control (9) B cells by treatment with CD40L, but little changed by the addition of anti-Ig or by the addition of P/I after CD40L stimulation (Fig. 6GoB). This was also true for Btk-deficient B cells, and thus, induction of Fas resistance in the absence of Btk cannot be explained by shifts in Fas expression. Notably in this experiment, CD40L-stimulated Btk-deficient B cells stained less intensely for Fas (mean fluorescence intensity = 137) than CD40L-stimulated control B cells (mean fluorescence intensity = 191), and yet were more sensitive to Fas-mediated apoptosis, demonstrating that although Fas expression is necessary for Fas killing, there is not always a strict correlation between Fas levels and Fas sensitivity.



View larger version (24K):
[in this window]
[in a new window]
  		FIGURE 5. sIg signaling induces resistance to sFasL in Btk-deficient B cells. Primary splenic B cells obtained from Btk-deficient (Btk KO) or WT control (WT) mice were cultured with CD40L/CD8{alpha} fusion protein cross-linked with anti-CD8 Ab for 48 h (CD40L), or with CD40L for 48 h plus either F(ab')2 fragments of goat anti-mouse IgM at 10 µg/ml (CD40L/{alpha}Ig), or the combination of PMA at 100 ng/ml and ionomycin at 600 ng/ml (CD40L/P + I), added during the last 24 h of culture, as indicated. B cells were then radiolabeled and tested as targets for Fas-dependent cytotoxicity mediated by sFasL (50 ng/ml) in standard 51Cr release assays. The means of specific lysis for three independent experiments are shown, along with lines indicating the SEM.

 


View larger version (39K):
[in this window]
[in a new window]
  		FIGURE 6. sIg signaling induces resistance in Btk-deficient (Btk KO) B cells at various doses of sFasL. Primary splenic B cells obtained from Btk-deficient (Btk KO) or WT control (WT) mice were treated as described in the legend to Fig. 5Go. Separate aliquots of B cells were radiolabeled and tested as targets for susceptibility to Fas-dependent cytotoxicity at the doses of sFasL indicated, and were stained with FITC-labeled Jo-2 monoclonal anti-Fas Ab and analyzed for Fas expression by flow cytometry. A, Mean percentage of specific cell lysis of triplicate assays for each condition, along with a line indicating the SEM. B, Relative cell number as a function of fluorescence intensity for each condition. The results of a single representative experiment are shown.

 
To clarify the influence of Btk on Fas sensitivity, the susceptibility to FasL-mediated cytotoxicity of CD40L-stimulated xid, Btk-deficient, and control B cells were compared. To relate independent experiments and avoid saturating levels of FasL that might induce maximal chromium release in all B cells (thereby obscuring intrinsic differences), cytotoxicity was evaluated for control B cells at the dose of sFasL that produced specific cell lysis nearest to 50% in xid and in Btk-deficient B cells. Results are shown in Fig. 7Go. The susceptibility of xid and control B cells to Fas killing after stimulation by CD40L did not differ (n = 4). In contrast, the Fas sensitivity of CD40L-stimulated Btk-deficient B cells exceeded that of CD40L-stimulated control B cells by a significant margin (Fig. 5Go and data not shown; n = 4, p < 0.03 by Student’s t test), as suggested by the data displayed in Figs. 5Go and 6Go. The Fas sensitivity of stimulated Btk-deficient and control B cells also differed when cytotoxicity was induced by FasL-bearing Th1 effector cells. In this case, specific cell lysis was 51 ± 2.2 vs 38 ± 2.3, for Btk-deficient and control B cells, respectively (Fig. 4Go and data not shown; n = 6, p < 0.03). Pooled data yielded values of 51 ± 1.5 and 36 ± 2.5, respectively (n = 10, p < 0.001). Therefore, regardless of the Fas trigger, Btk-deficient B cells appear to undergo apoptosis more readily and/or more extensively at a given level of Fas engagement.



View larger version (31K):
[in this window]
[in a new window]
  		FIGURE 7. Btk-deficient B cells display enhanced sensitivity to FasL-induced apoptosis after treatment with CD40L alone. Primary splenic B cells obtained from xid (Xid), Btk-deficient (Btk KO), or WT control (WT, CBA, and C57BL/6, respectively) mice were cultured with CD40L/CD8{alpha} fusion protein cross-linked with anti-CD8 Ab for 48 h (CD40L), and then radiolabeled and tested as targets for Fas-dependent cytotoxicity mediated by sFasL in standard 51Cr release assays. In each experiment, the dose of sFasL that produced specific lysis nearest 50% in xid or in Btk-deficient B cells was noted, and the specific lysis at that dose compared for Btk mutant/deficient B cells and appropriate control B cells. The means of specific lysis for four independent experiments for xid B cells and four independent experiments for Btk-deficient B cells are shown, along with lines indicating the SEM. The doses of sFasL represented range from 50 to 60 ng/ml for xid and control B cells, and 15–60 ng/ml for Btk-deficient B cells and controls.

 
Expression of Bcl-2 family members in Btk-deficient B cells

Bcl-xL has been implicated as a terminal mediator of sIg-induced Fas resistance, as noted earlier. However, it has been reported that sIg engagement fails to induce Bcl-xL in xid B cells (49, 50, 51). This raises the question of whether the same would be found to be true when anti-Ig is added to CD40L-stimulated B cells. To address this issue, xid and control B cells were stimulated with anti-Ig alone, or were stimulated for 48 h with CD40L in combination with anti-Ig added for the last few hours of culture, after which RNA was obtained and evaluated for expression of Bcl-2 family members by RNase protection assay. Results are shown in Fig. 8Go. Anti-Ig-induced up-regulation of Bcl-xL and of Bfl-1 was blocked by Btk mutation. However, after treatment with CD40L, anti-Ig produced similar levels of Bfl-1 and Bcl-xL expression in xid and control B cells; this was particularly evident for Bfl-1. Therefore, prior CD40L stimulation appears to overcome or circumvent the interruption in sIg signaling imposed by mutation of Btk, at least insofar as the outcomes of Bfl-1 and Bcl-xL expression are concerned. In these experiments, Bcl-2 and Bak were not induced by any of the treatments, as previously observed in our unpublished observations of primary B cell RNA by RT-PCR. Bad, Bax, and Bcl-w were not detected.



View larger version (81K):
[in this window]
[in a new window]
  		FIGURE 8. Bcl-xL and Bfl-1 are similarly induced in xid B cells and in control B cells. Primary splenic B cells obtained from xid (Xid) or WT control (WT) mice were cultured with CD40L/CD8{alpha} fusion protein cross-linked with anti-CD8 Ab (CD40L) for 48 h alone, or CD40L plus F(ab')2 fragments of goat anti-mouse IgM at 10 µg/ml ({alpha}IgM) for the last two and the last 6 h of culture as indicated. Naive xid and control B cells were also stimulated by anti-IgM alone ({alpha}IgM) for 4 h. RNA was prepared and DNase treated. RNase protection was conducted as described in Materials and Methods; fragments were size separated by urea-PAGE and quantitated by phosphorimager analysis. Top, Results from one of three comparable experiments. Bottom, Mean values for pixel density per square millimeter of Bfl-1 (BFL-1) and Bcl-xL (BCL-X) expression normalized to expression of L32 for three independent experiments along with lines indicating the SEM.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The work presented in this study demonstrates that Ag receptor engagement induces resistance to Fas-mediated apoptosis in primary B cells in the face of loss or mutation of Btk. sIg signaling for S phase entry and for Bcl-xL expression fails in the absence of functioning Btk, and in the present experiments xid and Btk-deficient B cells did not incorporate thymidine (data not shown), nor up-regulate Bcl-xL gene (Fig. 8Go) or protein (data not shown) expression after stimulation with anti-Ig. Thus, the intracellular signaling pathway responsible for sIg-induced Fas resistance must differ in important ways from the pathway(s) responsible for cell cycle progression and Bcl-xL (and Bfl-1) expression. Moreover, these results indicate that Btk is not required for induction of Fas resistance produced by sIg signaling in primary murine B cells, notwithstanding reports that Btk binds Fas and interferes with apoptosis in DT40 cells (42).

CD40 engagement alone produced up-regulation of Fas expression and acquisition of sensitivity to Fas-mediated apoptosis, despite reports that CD40-triggered proliferative responses are blocked or reduced in xid and Btk KO B cells (41, 52, 53). These apoptotic-related outcomes represent new additions to signaling events known to be preserved under conditions of Btk mutation and/or deficiency, such as up-regulation of CD23 (54). Notably, the complete loss of Btk was associated with significantly increased Fas sensitivity in B cells stimulated by CD40L alone. However, the relative enhancement of susceptibility to Fas signaling for cell death in murine B cells (Fig. 7Go) is much reduced in comparison to that previously reported for Btk-deficient DT40 cells (42); regardless, these findings support the notion that Btk plays a role in establishing the level of sensitivity to Fas killing of activated mammalian B cells, even if this role is of less consequence than that reported for chicken B cells. In contrast, the xid mutation was not associated with any alteration in the susceptibility of CD40L-stimulated B cells to Fas engagement by sFasL or by Th1 cells, suggesting that a region other than the pleckstrin homology domain is responsible for the influence of Btk on Fas-mediated apoptosis (28, 29, 55).

Fas resistance was evident in B cells treated with CD40L plus anti-Ig even at higher doses of sFasL that produced substantial levels of cytotoxicity (Figs. 5Go and 6Go). Although the amount of specific cell lysis that was reversed was similar at higher and lower doses of sFasL, the proportion of apoptosing B cells that were protected declined. This may suggest that higher doses of sFasL recruit B cells into the apoptotic pathway that are incapable of being rescued by sIg engagement. However, Fas resistance is relative, not absolute, and cannot be detected in the face of very strong Fas signaling such as that produced by Jo-2 monoclonal anti-Fas Ab (13). Thus, the loss of proportionate Fas resistance at higher doses of sFasL may reflect the balance between the intensity of pro- and anti-apoptotic signals initiated by Fas and Ag receptor engagement.

The capacity of Bcl-2 family members to oppose Fas-mediated apoptosis in lymphocytes remains controversial (19, 56, 57, 58, 59). Some of the discrepancy in previous results may relate to the use of cell lines which rely to different extents on mitochondrial changes and cytochrome c release as intermediates in Fas signaling for cell death (57). In previous work from this laboratory with transgenic mice, Bcl-xL was implicated as a terminal effector of sIg-induced Fas resistance (19). However, in recent work by Liou and colleagues (60) with c-Rel-deficient B cells, Fas resistance was observed in the absence of substantial Bcl-xL induction. Although it had been reported that sIg signaling in naive xid B cells failed to induce Bcl-xL (Refs. 49, 50, 51 ; Fig. 8Go), the present study specifically probed the expression of Bcl-xL and other Bcl-2 family members in B cells following stimulation by an optimal Fas resistance-inducing regimen, encompassing pretreatment with CD40L and sequential treatment with anti-Ig. Surprisingly, both Bfl-1 and Bcl-xL were up-regulated to the same extent in xid and control B cells when anti-Ig stimulation followed CD40L treatment, even though neither were induced in naive xid B cells by anti-Ig treatment alone. This suggests the possibility that Bfl-1, Bcl-xL, or both may participate in sIg-induced Fas resistance, although neither is induced by anti-Ig in untreated B cells. More importantly, these results suggest that sIg signaling is altered by prior B cell stimulation with CD40L such that an outcome precluded by Btk mutation (e.g., Bfl-1 expression) is now permitted. Elucidation of the sIg signaling pathway that is enabled by prior treatment with CD40L is likely to provide insight into the mechanism by which sIg mediates Fas resistance. The observation that Bfl-1 expression depends on NF-{kappa}B (61, 62, 63, 64) suggests a potential mediator that may be involved.


   Footnotes
 
1 This work was supported by U.S. Public Health Service Grants T32 AI07309, T32 HL07501, AI41054 (to R.T.W.), AI40181 (to T.L.R.), and AI45112 (to T.L.R.) awarded by the National Institutes of Health. Back

2 J.R.T. and R.S.N. contributed equally to this work. Back

3 Current address: Cancer Biomarkers Research Group, Division of Cancer Prevention, National Cancer Institute, Rockville, MD 20852. Back

4 Current address: Center for Blood Research, Harvard Medical School, Longwood Medical Research Center Room 501, 221 Longwood Avenue, Boston, MA 02115. Back

5 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

6 Abbreviations used in this paper: sIg, surface Ig; Btk, Bruton’s tyrosine kinase; xid, X-linked immunodeficiency; CD40L, CD40 ligand; FasL, Fas ligand; WT, wild type; P/I, PMA plus ionomycin; KO, knockout; DT, diphtheria toxoid; sFasL, soluble FasL. Back

Received for publication February 2, 2001. Accepted for publication January 11, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Watanabe-Fukunaga, R., C. I. Brannan, N. G. Copeland, N. A. Jenkins, S. Nagata. 1992. Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis. Nature 356:314.[Medline]
  2. Fisher, G. H., F. J. Rosenberg, S. E. Straus, J. K. Dale, L. A. Middleton, A. Y. Lin, W. Strober, M. J. Lenardo, J. M. Puck. 1995. Dominant interfering Fas gene mutations impair apoptosis in a human autoimmune lymphoproliferative syndrome. Cell 81:935.[Medline]
  3. Rieux-Laucat, F., F. Le Deist, C. Hivroz, I. A. Roberts, K. M. Debatin, A. Fischer, J. P. de Villartay. 1995. Mutations in Fas associated with human lymphoproliferative syndrome and autoimmunity. Science 268:1347.[Abstract/Free Full Text]
  4. Perkins, D. L., J. Michaelson, R. M. Glaser, A. Marshak-Rothstein. 1987. Selective elimination of non-lpr lymphoid cells in mice undergoing lpr-mediated graft-vs-host disease. J. Immunol. 139:1406.[Abstract]
  5. Nemazee, D., C. Guiet, K. Buerki, A. Marshak-Rothstein. 1991. B lymphocytes from the autoimmune-prone mouse strain MLR/lpr manifest an intrinsic defect in tetraparental MRL/lpr in equilibrium DBA/2 chimeras. J. Immunol. 147:2536.[Abstract/Free Full Text]
  6. Sobel, E. S., V. N. Kakkanaiah, J. Schiffenbauer, E. A. Reap, P. L. Cohen, R. A. Eisenberg. 1998. Novel immunoregulatory B cell pathways revealed by lpr-/+ mixed chimeras. J. Immunol. 160:1497.[Abstract/Free Full Text]
  7. Fukuyama, H., M. Adachi, S. Suematsu, K. Miwa, T. Suda, N. Yoshida, S. Nagata. 1998. Transgenic expression of Fas in T cells blocks lymphoproliferation but not autoimmune disease in MRL-lpr mice. J. Immunol. 160:3805.[Abstract/Free Full Text]
  8. Rathmell, J. C., M. P. Cooke, W. Y. Ho, J. Grein, S. E. Townsend, M. M. Davis, C. C. Goodnow. 1995. CD95 (Fas)-dependent elimination of self-reactive B cells upon interaction with CD4+ T cells. Nature 376:181.[Medline]
  9. Rothstein, T. L., J. K. Wang, D. J. Panka, L. C. Foote, Z. Wang, B. Stanger, H. Cui, S. T. Ju, A. Marshak-Rothstein. 1995. Protection against Fas-dependent Th1-mediated apoptosis by antigen receptor engagement in B cells. Nature 374:163.[Medline]
  10. Schattner, E. J., K. B. Elkon, D. H. Yoo, J. Tumang, P. H. Krammer, M. K. Crow, S. M. Friedman. 1995. CD40 ligation induces Apo-1/Fas expression on human B lymphocytes and facilitates apoptosis through the Apo-1/Fas pathway. J. Exp. Med. 182:1557.[Abstract/Free Full Text]
  11. Garrone, P., E. M. Neidhardt, E. Garcia, L. Galibert, C. van Kooten, J. Banchereau. 1995. Fas ligation induces apoptosis of CD40-activated human B lymphocytes. J. Exp. Med. 182:1265.[Abstract/Free Full Text]
  12. Foote, L. C., R. G. Howard, A. Marshak-Rothstein, T. L. Rothstein. 1996. IL-4 induces Fas resistance in B cells. J. Immunol. 157:2749.[Abstract]
  13. Foote, L. C., T. J. Schneider, G. M. Fischer, J. K. Wang, B. Rasmussen, K. A. Campbell, D. H. Lynch, S. T. Ju, A. Marshak-Rothstein, T. L. Rothstein. 1996. Intracellular signaling for inducible antigen receptor-mediated Fas resistance in B cells. J. Immunol. 157:1878.[Abstract]
  14. Rathmell, J. C., S. E. Townsend, J. C. Xu, R. A. Flavell, C. C. Goodnow. 1996. Expansion or elimination of B cells in vivo: dual roles for CD40- and Fas (CD95)-ligands modulated by the B cell antigen receptor. Cell 87:319.[Medline]
  15. Lagresle, C., P. Mondiere, C. Bella, P. H. Krammer, T. Defrance. 1996. Concurrent engagement of CD40 and the antigen receptor protects naive and memory human B cells from APO-1/Fas-mediated apoptosis. J. Exp. Med. 183:1377.[Abstract/Free Full Text]
  16. Ozdemirli, M., M. El-Khatib, L. C. Foote, J. K. Wang, A. Marshak-Rothstein, T. L. Rothstein, S. T. Ju. 1996. Fas (CD95)/Fas ligand interactions regulate antigen-specific, major histocompatibility complex-restricted T/B cell proliferative responses. Eur. J. Immunol. 26:415.[Medline]
  17. Wang, J. K. M., S. T. Ju, A. Marshak-Rothstein. 2000. Protection of T cells from activation-induced cell death by Fas+ B cells. Eur. J. Immunol. 3:931.
  18. Foote, L. C., A. Marshak-Rothstein, T. L. Rothstein. 1998. Tolerant B lymphocytes acquire resistance to Fas-mediated apoptosis after treatment with interleukin 4 but not after treatment with specific antigen unless a surface immunoglobulin threshold is exceeded. J. Exp. Med. 187:847.[Abstract/Free Full Text]
  19. Schneider, T. J., D. Grillot, L. C. Foote, G. E. Nunez, T. L. Rothstein. 1997. Bcl-x protects primary B cells against Fas-mediated apoptosis. J. Immunol. 159:4834.[Abstract]
  20. Schneider, T. J., G. M. Fischer, T. J. Donohoe, T. P. Colarusso, T. L. Rothstein. 1999. A novel gene coding for a Fas apoptosis inhibitory molecule (FAIM) isolated from inducibly Fas-resistant B lymphocytes. J. Exp. Med. 189:949.[Abstract/Free Full Text]
  21. Van Parijs, L., Y. Refaeli, A. K. Abbas, D. Baltimore. 1999. Autoimmunity as a consequence of retrovirus-mediated expression of c-FLIP in lymphocytes. Immunity 11:763.[Medline]
  22. Wang, J., A. A. Lobito, F. Shen, F. Hornung, A. Winoto, M. J. Lenardo. 2000. Inhibition of Fas-mediated apoptosis by the B cell antigen receptor through c-FLIP. Eur. J. Immunol. 30:155.[Medline]
  23. Yang, W. C., Y. Collette, J. A. Nunes, D. Olive. 2000. Tec kinases: a family with multiple roles in immunity. Immunity 12:373.[Medline]
  24. de Weers, M., M. C. Verschuren, M. E. Kraakman, R. G. Mensink, R. K. Schuurman, J. J. van Dongen, R. W. Hendriks. 1993. The Bruton’s tyrosine kinase gene is expressed throughout B cell differentiation, from early precursor B cell stages preceding immunoglobulin gene rearrangement up to mature B cell stages. Eur. J. Immunol. 23:3109.[Medline]
  25. Smith, C. I., B. Baskin, P. Humire-Greiff, J. N. Zhou, P. G. Olsson, H. S. Maniar, P. Kjellen, J. D. Lambris, B. Christensson, L. Hammarstrom, et al 1994. Expression of Bruton’s agammaglobulinemia tyrosine kinase gene, BTK, is selectively down-regulated in T lymphocytes and plasma cells. J. Immunol. 152:557.[Abstract]
  26. Vetrie, D., I. Vorechovsky, P. Sideras, J. Holland, A. Davies, F. Flinter, L. Hammarstrom, C. Kinnon, R. Levinsky, M. Bobrow, et al 1993. The gene involved in X-linked agammaglobulinaemia is a member of the src family of protein-tyrosine kinases. Nature 361:226.[Medline]
  27. Tsukada, S., D. C. Saffran, D. J. Rawlings, O. Parolini, R. C. Allen, I. Klisak, R. S. Sparkes, H. Kubagawa, T. Mohandas, S. Quan, et al 1993. Deficient expression of a B cell cytoplasmic tyrosine kinase in human X-linked agammaglobulinemia. Cell 72:279.[Medline]
  28. Rawlings, D. J., D. C. Saffran, S. Tsukada, D. A. Largaespada, J. C. Grimaldi, L. Cohen, R. N. Mohr, J. F. Bazan, M. Howard, N. G. Copeland, et al 1993. Mutation of unique region of Bruton’s tyrosine kinase in immunodeficient XID mice. Science 261:358.[Abstract/Free Full Text]
  29. Thomas, J. D., P. Sideras, C. I. Smith, I. Vorechovsky, V. Chapman, W. E. Paul. 1993. Colocalization of X-linked agammaglobulinemia and X-linked immunodeficiency genes. Science 261:355.[Abstract/Free Full Text]
  30. Mahajan, S., J. Fargnoli, A. L. Burkhardt, S. A. Kut, S. J. Saouaf, J. B. Bolen. 1995. Src family protein tyrosine kinases induce autoactivation of Bruton’s tyrosine kinase. Mol. Cell Biol. 15:5304.[Abstract]
  31. Afar, D. E., H. Park, B. W. Howell, D. J. Rawlings, J. Cooper, O. N. Witte. 1996. Regulation of Btk by Src family tyrosine kinases. Mol. Cell Biol. 16:3465.[Abstract]
  32. Salim, K., M. J. Bottomley, E. Querfurth, M. J. Zvelebil, I. Gout, R. Scaife, R. L. Margolis, R. Gigg, C. I. Smith, P. C. Driscoll, M. D. Waterfield, G. Panayotou. 1996. Distinct specificity in the recognition of phosphoinositides by the pleckstrin homology domains of dynamin and Bruton’s tyrosine kinase. EMBO J. 15:6241.[Medline]
  33. Li, Z., M. I. Wahl, A. Eguinoa, L. R. Stephens, P. T. Hawkins, O. N. Witte. 1997. Phosphatidylinositol 3-kinase-{gamma} activates Bruton’s tyrosine kinase in concert with Src family kinases. Proc. Natl. Acad. Sci. USA 94:13820.[Abstract/Free Full Text]
  34. Satterthwaite, A. B., C. A. Lowell, W. N. Khan, P. Sideras, F. W. Alt, O. N. Witte. 1998. Independent and opposing roles for Btk and lyn in B and myeloid signaling pathways. J. Exp. Med. 188:833.[Abstract/Free Full Text]
  35. Suzuki, H., Y. Terauchi, M. Fujiwara, S. Aizawa, Y. Yazaki, T. Kadowaki, S. Koyasu. 1999. Xid-like immunodeficiency in mice with disruption of the p85{alpha} subunit of phosphoinositide 3-kinase. Science 283:390.[Abstract/Free Full Text]
  36. Fruman, D. A., S. B. Snapper, C. M. Yballe, L. Davidson, J. Y. Yu, F. W. Alt, L. C. Cantley. 1999. Impaired B cell development and proliferation in absence of phosphoinositide 3-kinase p85{alpha}. Science 283:393.[Abstract/Free Full Text]
  37. Takata, M., T. Kurosaki. 1996. A role for Bruton’s tyrosine kinase in B cell antigen receptor-mediated activation of phospholipase C-{gamma}2. J. Exp. Med. 184:31.[Abstract/Free Full Text]
  38. Fluckiger, A. C., Z. Li, R. M. Kato, M. I. Wahl, H. D. Ochs, R. Longnecker, J. P. Kinet, O. N. Witte, A. M. Scharenberg, D. J. Rawlings. 1998. Btk/Tec kinases regulate sustained increases in intracellular Ca2+ following B-cell receptor activation. EMBO J. 17:1973.[Medline]
  39. Rigley, K. P., M. M. Harnett, R. J. Phillips, G. G. Klaus. 1989. Analysis of signaling via surface immunoglobulin receptors on B cells from CBA/N mice. Eur. J. Immunol. 19:2081.[Medline]
  40. Sieckmann, D. G., R. Asofsky, D. E. Mosier, I. M. Zitron, W. E. Paul. 1978. Activation of mouse lymphocytes by anti-immunoglobulin. I. Parameters of the proliferative response. J. Exp. Med. 147:814.[Abstract/Free Full Text]
  41. Khan, W. N., F. W. Alt, R. M. Gerstein, B. A. Malynn, I. Larsson, G. Rathbun, L. Davidson, S. Muller, A. B. Kantor, L. A. Herzenberg, et al 1995. Defective B cell development and function in Btk-deficient mice. Immunity 3:283.[Medline]
  42. Vassilev, A., Z. Ozer, C. Navara, S. Mahajan, F. M. Uckun. 1999. Bruton’s tyrosine kinase as an inhibitor of the Fas/CD95 death-inducing signaling complex. J. Biol. Chem. 274:1646.[Abstract/Free Full Text]
  43. Nisitani, S., A. B. Satterthwaite, K. Akashi, I. L. Weissman, O. N. Witte, M. I. Wahl. 2000. Posttranscriptional regulation of Bruton’s tyrosine kinase expression in antigen receptor-stimulated splenic B cells. Proc. Natl. Acad. Sci. USA 97:2737.[Abstract/Free Full Text]
  44. Lane, P., T. Brocker, S. Hubele, E. Padovan, A. Lanzavecchia, F. McConnell. 1993. Soluble CD40 ligand can replace the normal T cell-derived CD40 ligand signal to B cells in T cell-dependent activation. J. Exp. Med. 177:1209.[Abstract/Free Full Text]
  45. Francis, D. A., J. G. Karras, X. Y. Ke, R. Sen, T. L. Rothstein. 1995. Induction of the transcription factors NF-{kappa}B, AP-1 and NF-AT during B cell stimulation through the CD40 receptor. Int. Immunol. 7:151.[Abstract/Free Full Text]
  46. Rothstein, T. L., T. R. Baeker, R. A. Miller, D. L. Kolber. 1986. Stimulation of murine B cells by the combination of calcium ionophore plus phorbol ester. Cell Immunol. 102:364.[Medline]
  47. Woodland, R. T., M. R. Schmidt, J. E. Riggs, S. J. Korsmeyer, A. M. Lussier, K. A. Gravel. 1995. Radiation-induced apoptosis is differentially regulated in primary B cells from normal mice and mice with the CBA/N X-linked immunodeficiency. J. Immunol. 155:3453.[Abstract]
  48. Bajpai, U. D., K. Zhang, M. Teutsch, R. Sen, H. H. Wortis. 2000. Bruton’s tyrosine kinase links the B cell receptor to nuclear factor {kappa}B activation. J. Exp. Med. 191:1735.[Abstract/Free Full Text]
  49. Choi, M. S., M. Holmann, C. J. Atkins, G. G. Klaus. 1996. Expression of blc-x during mouse B cell differentiation and following activation by various stimuli. Eur. J. Immunol. 26:676.[Medline]
  50. Anderson, J. S., M. Teutsch, Z. Dong, H. H. Wortis. 1996. An essential role for Bruton’s tyrosine kinase in the regulation of B-cell apoptosis. Proc. Natl. Acad. Sci. USA 93:10966.[Abstract/Free Full Text]
  51. Solvason, N., W. W. Wu, N. Kabra, F. Lund-Johansen, M. G. Roncarolo, T. W. Behrens, D. A. Grillot, G. Nunez, E. Lees, M. Howard. 1998. Transgene expression of Bcl-xL permits anti-immunoglobulin (Ig)-induced proliferation in xid B cells. J. Exp. Med. 187:1081.[Abstract/Free Full Text]
  52. Hasbold, J., G. G. Klaus. 1994. B cells from CBA/N mice do not proliferate following ligation of CD40. Eur. J. Immunol. 24:152.[Medline]
  53. Baba, M., Y. Kikuchi, S. Mori, H. Kimoto, S. Inui, N. Sakaguchi, J. Inoue, T. Yamamoto, T. Takemori, M. Howard, K. Takatsu. 1997. Mouse germinal center B cells with the xid mutation retain responsiveness to antimouse CD40 antibodies but diminish IL-5 responsiveness. Int. Immunol. 9:1463.[Abstract/Free Full Text]
  54. Nonoyama, S., S. Tsukada, T. Yamadori, T. Miyawaki, Y. Z. Jin, C. Watanabe, T. Morio, J. Yata, H. D. Ochs. 1998. Functional analysis of peripheral blood B cells in patients with X-linked agammaglobulinemia. J. Immunol. 161:3925.[Abstract/Free Full Text]
  55. Shaw, G.. 1993. Identification of novel pleckstrin homology (PH) domains provides a hypothesis for PH domain function. Biochim. Biophys. Acta 195:1145.
  56. Strasser, A., A. W. Harris, D. C. Huang, P. H. Krammer, S. Cory. 1995. Bcl-2 and Fas/APO-1 regulate distinct pathways to lymphocyte apoptosis. EMBO J. 14:6136.[Medline]
  57. Scaffidi, C., S. Fulda, A. Srinivasan, C. Friesen, F. Li, K. J. Tomaselli, K. M. Debatin, P. H. Krammer, M. E. Peter. 1998. Two CD95 (APO-1/Fas) signaling pathways. EMBO J. 17:1675.[Medline]
  58. Schmitz, I., H. Walczak, P. H. Krammer, M. E. Peter. 2000. The two CD95 apoptosis signaling pathways may be a way of cells to respond to different amounts and/or forms of CD95 ligand produced in different tissues. Cell Death Differ. 7:756.
  59. Huang, D. C. S., J. Tschopp, A. Strasser. 2000. Bcl-2 does not inhibit cell death induced by the physiological Fas ligand: implications for the existence of type I and type II cells. Cell Death Differ. 7:754.[Medline]
  60. Owyang, A. M., J. R. Tumang, B. R. Schram, C. Y. Hsia, T. W. Behrens, T. L. Rothstein, H. C. Liou. 2001. c-Rel is required for the protection of B cells from antigen receptor-mediated, but not Fas-mediated, apoptosis. J. Immunol. 167:4948.[Abstract/Free Full Text]
  61. Zong, W. X., L. C. Edelstein, C. Chen, J. Bash, C. Gelinas. 1999. The prosurvival Bcl-2 homolog Bfl-1/A1 is a direct transcriptional target of NF-{kappa}B that blocks TNF{alpha}-induced apoptosis. Genes Dev. 13:382.[Abstract/Free Full Text]
  62. Grumont, R. J., I. J. Rourke, S. Gerondakis. 1999. Rel-dependent induction of A1 transcription is required to protect B cells from antigen receptor ligation-induced apoptosis. Genes Dev. 13:400.[Abstract/Free Full Text]
  63. Lee, H. H., H. Dadgostar, Q. Cheng, J. Shu, G. Cheng. 1999. NF-{kappa}B-mediated up-regulation of Bcl-x and Bfl-1/A1 is required for CD40 survival signaling in B lymphocytes. Proc. Natl. Acad. Sci. USA 96:9136.[Abstract/Free Full Text]
  64. Wang, C. Y., D. C. Guttridge, M. W. Mayo, Jr A. S. Baldwin. 1999. NF-{kappa}B induces expression of the Bcl-2 homologue A1/Bfl-1 to preferentially suppress chemotherapy-induced apoptosis. Mol. Cell Biol. 19:5923.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Int ImmunolHome page
B. Alabyev, R. Vuyyuru, and T. Manser
Influence of Fas on the regulation of the response of an anti-nuclear antigen B cell clonotype to foreign antigen
Int. Immunol., October 1, 2008; 20(10): 1279 - 1287.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
T. Mizuno and T. L. Rothstein
B Cell Receptor (BCR) Cross-Talk: CD40 Engagement Creates an Alternate Pathway for BCR Signaling That Activates I{kappa}B Kinase/I{kappa}B{alpha}/NF-{kappa}B without the Need for PI3K and Phospholipase C{gamma}
J. Immunol., May 15, 2005; 174(10): 6062 - 6070.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
T. Mizuno and T. L. Rothstein
B Cell Receptor (BCR) Cross-Talk: CD40 Engagement Enhances BCR-Induced ERK Activation
J. Immunol., March 15, 2005; 174(6): 3369 - 3376.
[Abstract] [Full Text] [PDF]

Home page
 JEMHome page
U. Schmidt, E. van den Akker, M. Parren-van Amelsvoort, G. Litos, M. de Bruijn, L. Gutierrez, R. W. Hendriks, W. Ellmeier, B. Lowenberg, H. Beug, et al.
Btk Is Required for an Efficient Response to Erythropoietin and for SCF-controlled Protection against TRAIL in Erythroid Progenitors
J. Exp. Med., March 15, 2004; 199(6): 785 - 795.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
T. Mizuno and T. L. Rothstein
Cutting Edge: CD40 Engagement Eliminates the Need for Bruton's Tyrosine Kinase in B Cell Receptor Signaling for NF-{kappa}B
J. Immunol., March 15, 2003; 170(6): 2806 - 2810.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
B. R. Schram and T. L. Rothstein
NF-{kappa}B Is Required for Surface Ig-Induced Fas Resistance in B Cells
J. Immunol., March 15, 2003; 170(6): 3118 - 3124.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tumang, J. R.
Right arrow Articles by Rothstein, T. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tumang, J. R.
Right arrow Articles by Rothstein, T. L.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS