The JI PBL Intereron Source
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
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Catlett, I. M.
Right arrow Articles by Bishop, G. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Catlett, I. M.
Right arrow Articles by Bishop, G. A.
The Journal of Immunology, 2001, 166: 6019-6024.
Copyright © 2001 by The American Association of Immunologists

Signaling Through MHC Class II Molecules Blocks CD95-Induced Apoptosis1

Ian M. Catlett*, Ping Xie*, Bruce S. Hostager* and Gail A. Bishop2,*,{dagger},{ddagger},§

Departments of * Microbiology and {dagger} Internal Medicine and {ddagger} Graduate Program in Immunology, University of Iowa, and § Veterans Affairs Medical Center, Iowa City, IA 52242


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
B cells are induced to express CD95 upon interaction with T cells. This interaction renders the B cells sensitive to CD95-mediated apoptosis, but ligation of proviability surface receptors is able to inhibit apoptosis induction. MHC class II is a key molecule required for Ag presentation to Th cells, productive T cell-B cell interaction, and B cell activation. We demonstrate here for the first time that MHC class II ligation also confers a rapid resistance to CD95-induced apoptosis, an affect that does not require de novo protein synthesis. Signaling through class II molecules blocks the activation of caspase 8, but does not affect the association of CD95 and Fas-associated death domain-containing protein. MHC class II ligation thus blocks proximal signaling events in the CD95-mediated apoptotic pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
How cells are induced to live or die by signals from their external environment is a question of continuing interest. Regulation of viability and programmed cell death is of particular importance to lymphocytes, because aberrant activation of immune responses may result in a state of autoimmunity. The immune system is regulated to avoid the production of cells that are autoreactive. The activation of B cells by CD40 ligation is accompanied by an increase in expression of CD95 and sensitivity to CD95-induced apoptosis (1), which is important for the elimination of autoreactive B cells (2, 3). However, B cells can be protected from CD95-mediated apoptosis by engagement of the B cell Ag receptor (BCR),3 CD40, or the IL-4 receptor (4, 5, 6), and mice transgenic for IL-4 receptor expression show enhanced autoimmunity (6).

Previously described mechanisms for blocking CD95 have involved the transcriptional activation of genes encoding antiapoptotic molecules. BCR ligation results in the increased production of antiapoptotic proteins such as bcl-xL, caspase 8 inhibitory protein, and Fas apoptosis inhibitory molecule (7, 8, 9, 10), while CD40 signaling can up-regulate bcl-xL and A20 (7, 11). Additionally, we have shown that BCR ligation leads to the immediate blocking of CD95-mediated apoptosis in a manner independent of de novo transcription (4). Thus, B cells receive different signals that may use distinct mechanisms to render them insensitive to CD95-mediated apoptosis.

The pathway used by CD95 to rapidly induce apoptosis in cells is initiated by the recruitment of the adapter molecule called Fas-associated death domain-containing protein (FADD), which in turn binds to caspase 8 and a molecule called caspase 8-associated huge protein (FLASH) (12, 13, 14, 15). Both FADD and caspase 8 are required for CD95-induced apoptosis (16, 17). Assembly of this complex leads to the autocatalytic cleavage and activation of caspase 8 (18, 19), which is then able to directly act upon downstream effector caspases such as caspase 3, 6, and 7 (20). Ligation of chimeric receptors that contain only caspase 8 as a cytoplasmic tail is sufficient to induce apoptosis (21).

A common theme linking several of the signals that counteract Fas-mediated apoptosis in B cells is that they are signals delivered to B cells during the course of Ag-specific activation events that require interactions with Th cells. Both B cell CD40 and IL-4 receptor signals are provided by interaction with activated T cells. However, such signals are not Ag specific, and thus could result in proviability signals delivered nonspecifically to B cells. We thus wondered whether a cognate signal, such as engagement of B cell class II, could also provide protection from Fas-mediated apoptosis, a protection that would be specific to B cells able to present Ag to the activated T cells. It is known that B cells respond to MHC class II ligation by increasing their adhesiveness, resulting in a closer interaction between the T and B cells (22). Additionally MHC class II ligation induces TNF secretion (23) and contributes to B cell differentiation as well as proliferation (24, 25, 26, 27). Thus, MHC class II can transduce signals that lead to the functional activation of B cells. Early events stimulated by MHC class II include the activation of protein tyrosine kinases, protein kinase C, phosphatidylinositol 3-kinase, and elevated intracellular calcium (28, 29). Ligation of MHC class II on murine B cells also activates adenylate cyclase and the production of cAMP (30, 31).

We have investigated whether MHC class II signaling is able to affect the induction of CD95-mediated apoptosis in murine B cells. We find that the simultaneous ligation of MHC class II and CD95 substantially reduced the induction of apoptosis when compared with CD95 ligation alone. This rescue event occurred rapidly and was independent of de novo protein synthesis. The loss of apoptosis was accompanied by a failure to activate caspase 8 by proteolytic cleavage, although CD95-FADD association remained intact.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and mice

The mouse B cell lines A20-2J (provided by David McKean, Mayo Medical Center, Rochester, MN) and CH12.LX (24) were maintained in RPMI 1640 supplemented with 10% FCS (Gemini, Calabasas, CA), 10 µM 2-ME, 2 µM glutamine (Sigma, St. Louis, MO), and antibiotics (B cell medium (BCM)). Splenocytes were prepared from BALB/c mice as described elsewhere (32). Briefly, mouse spleens were macerated with glass slides to obtain a single-cell suspension. The suspension was depleted of T cells by coating the cells with anti-Thy-1 (HO13.4; American Type Culture Collection, Manassas, VA) and treatment with complement. For some experiments, the B cells were then separated by centrifugation through a Percoll (Amersham Pharmacia Biotech, Piscataway NJ) step gradient. Low-density cells were collected from the 50 to 60% interface, medium-density cells from the 60 to 70% interface, and high-density cells from the 70 to 75% interface. Low-, medium-, and high-density cells were cultured for 24 h in the presence of anti-mouse CD40 mAb (see below) at 10 µg/ml. Surface expression of CD95 was confirmed by flow cytometry using a FACScan benchtop laser flow cytometer (Becton Dickinson, San Jose, CA). Splenocytes were washed twice with medium before secondary stimulation.

Antibodies

Hybridomas 14-4-4S, 17-3-3S (anti-Ek,d), OKT8 (anti-CD8; used as an isotype control for anti-class II), and UC8-169 (hamster IgG anti-2,4,6-trinitrophenyl, used as an isotype control for Jo-2) were purchased from the American Type Culture Collection. The anti-CD40 hybridoma 1C10 was a kind gift from Frances Lund (Trudeau Institute, Saranac Lake, NY) (33). Abs were harvested from clarified culture supernatant by saturated ammonium sulfate purification and quantitated by isotype-specific ELISA. Anti-CD95 (Jo-2) was purchased from PharMingen (San Diego, CA). Polyclonal rabbit anti-FADD antiserum (13) was obtained from Astar Winoto (University of California, Berkeley, CA). Rabbit polyclonal anti-caspase 8 (H-134, catalogue no. sc7890) and rabbit polyclonal anti-CD95 (sc-1024) Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Donkey anti-rabbit IgG conjugated to HRP was purchased from Jackson ImmunoResearch (West Grove, PA).

Cell stimulation

For all experiments shown, a combination of 14-4-4S and 17-3-3S mAbs was used at 5 µg/ml each to stimulate through I-E. The OKT8 isotype control Ab was used at 10 µg/ml. Stimulation through CD95 was accomplished with 100 ng/ml anti-CD95 (the same concentration was used for isotype control UC8-169). Cell concentrations and timing of stimuli are detailed below and in the figure legends for each type of experiment. For experiments presented in Fig. 3Go, cells were preincubated with 0.5–2.5 µM cycloheximide (CHX; Sigma) 30 min before addition of stimuli.



View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 3. MHC class II-mediated rescue was independent of de novo protein synthesis. A20 (A), CH12.LX (B), or normal splenic B cells (C) were pretreated with or without CHX for 30 min before 8 h (cell lines) or 16 h (splenic B cells) of stimulation, using mAbs as in Fig. 1Go. Treatments were: {square}, BCM only; {blacksquare}, anti-Fas; , anti-Fas + anti-class II; , anti-Fas + isotype control mAb. Apoptotic cells were quantitated as in Fig. 1Go. CHX was used at concentrations that inhibited de novo protein synthesis by 85–95% in each of the three cell types during the course of the experiments. The average ± SE of three experiments is shown.

 
Apoptosis assay

Apoptotic cells with subdiploid nuclei were detected as described previously (4). Briefly, cells at 5 x 105/ml were stimulated in 24-well plates as described above and 1 ml of culture was harvested and fixed with 900 µl of 70% ethanol. Samples were stored at -20°C until staining, then washed with 10 ml of PBS and resuspended in 500 µl of DNA extraction buffer (4 mM citric acid, 192 mM Na2HPO4, pH 7.8), for 5 min. The cells were pelleted and resuspended in 300 µl of staining buffer (PBS with 100 µg/ml RNase A and 10 µg/ml propidium iodide) for 30 min at room temperature. The cells were then analyzed on a benchtop flow cytometer (FACScan; Becton Dickinson) and subdiploid cells were counted.

Caspase 8 activity assay

Caspase 8 activity was determined by a fluorometric assay kit purchased from Clontech (Palo Alto, CA). Stimulations were as above except that 2 x 106 cells/ml were stimulated for 2–4 h. Cells were lysed in 240 µl of the provided buffer supplemented with complete protease inhibitor from Roche Molecular Biochemicals (Indianapolis, IN). The lysate was clarified by centrifugation for 5 min at 4°C and 16,500 x g. Sixty microliters of 2x reaction buffer supplemented with DTT and substrate, IETD-7-amino-4-trifluoromethyl coumarin (AMC), were added to 60 µl of lysate. The samples were incubated at 37°C for 1 h. Cleavage of the substrate results in the production of free AMC, which was excited at 400 nm, and fluorescence was read at 505 nm. Samples were read on a Spectronic Instruments Bowman Series 2 spectrophotometer (Rochester, NY).

Immunoprecipitation

Immunoprecipitations of CD95 were performed as described elsewhere (4, 13). Cells (2 x 107) were resuspended in 1 ml of medium and stimulated as described above for 10 or 90 min. The samples were then lysed in 1.4 ml of lysis buffer (0.5% Nonidet P-40, 50 mM Tris (pH 8.0), 150 mM NaCl, 1 mM EDTA, and 10 mM {beta}-glycerophosphate) and complete protease inhibitors (Boehringer Mannheim, Mannheim, Germany) for 30 min. Lysate was clarified by centrifugation for 30 min at 4°C and 16,500 x g. Fifty microliters were removed for a control blot to normalize lane loading. To the remaining lysate, 3 µg of anti-CD95 and 20 µl of protein G beads (Sigma) were added. The beads and lysate were tumbled for 1 h at 4°C and then washed 5x with 500 µl of lysis buffer. The immunoprecipitated proteins were separated on a 12% SDS-polyacrylamide gel and then transferred to nitrocellulose (Schleicher & Schuell, Keene, NH). The membrane was blocked with 10% milk and probed with anti-FADD (1:250) or anti-CD95 (1:250). The secondary Ab anti-rabbit IgG-HRP was detected by ECL (Pierce, Rockford, IL).

Western blotting

Cleavage of caspase 8 was detected by Western blotting, as described in our previous study (4). Cells (3 x 106) were stimulated for 3 h and then lysed in 60 µl of lysis buffer. The lysate was clarified by centrifugation at 16,500 x g at 4°C, and 10 µl of lysate was separated on a 10% SDS-polyacrylamide gel. Blotting for the p20 cleavage product of caspase 8 was performed as previously described (4) using the anti-caspase 8 Ab described above under Abs.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Murine B cells can be protected from CD95-mediated apoptosis by MHC class II ligation

To examine the effects of MHC class II ligation on CD95-mediated apoptosis, we used both normal splenic B cells and two B lymphoma-derived cell lines that are constitutively sensitive to CD95-mediated apoptosis. Normal splenic B cells were purified and separated based upon density as described in Materials and Methods. Because normal B cells express little to no detectable surface Fas, these cells were cultured for 24 h with 10 µg/ml anti-CD40 to induce CD95 expression and then washed twice in RPMI 1640 and restimulated with the indicated combinations of Abs. B cell lines were not prestimulated with anti-CD40. We observed no difference in the number of cells undergoing apoptosis when either left untreated or treated with anti-class II mAbs or an isotype control Ab for anti-class II (Fig. 1GoA). However, ligation of CD95 with agonistic Ab strongly induced apoptosis, and this apoptosis induction was blocked by simultaneous ligation of MHC class II. Replacement of anti-class II by an isotype control Ab resulted in the induction of apoptosis. Abs to I-A were also protective, and addition of blocking Ab to the FcR (24G2) did not alter the outcome of the experiments (data not shown).



View larger version (42K):
[in this window]
[in a new window]
  		FIGURE 1. Effect of MHC class II ligation on CD95-mediated apoptosis. A, B cells from BALB/c mice were prepared as described and stimulated for 24 h with anti-CD40 mAb to up-regulate Fas expression. Cells were then washed and restimulated for 16 h as indicated in Materials and Methods. The percentage of apoptotic cells was determined as described in Materials and Methods. Data presented are the mean ± SE of three experiments. B, A20 cells were treated with anti-CD95 + anti-class II, as indicated, for various periods of time. Apoptotic cells were quantitated as in A. A representative experiment of three performed is shown. Similar results were found using CH12.LX cells (data not shown).

 
The same observation can be made in the murine B cell lines A20-2J and CH12.LX. Both lymphomas express CD95 without requiring prior CD40 engagement and are sensitive to CD95-mediated apoptosis (4, 13). These cells were also specifically rescued from CD95-mediated apoptosis by simultaneous MHC class II ligation (Fig. 1GoB). Ligation of I-A MHC class II molecules was also able to rescue CH12.LX and A20 from CD95-mediated apoptosis (data not shown). A previous study of human B cell lines found that an anti-class II {alpha}-chain mAb promoted CD95-induced apoptosis in some but not all of the lines tested when given 30 min before anti-CD95 (34), so we also tested whether delaying addition of anti-CD95 for 30 min affected the outcome of our experiments. This delay did not decrease the effect of class II engagement on CD95-mediated apoptosis, and if we delayed addition of anti-Fas mAb for >1 h, class II-mediated rescue was somewhat enhanced (data not shown).

MHC class II ligation does not affect CD95 expression

One explanation for the inhibition of apoptosis could be that MHC class II ligation down-regulates the surface expression of CD95. To test this, cells were stimulated with Abs to MHC class II, CD40, or appropriate isotype controls for 24 h. Results are shown in Fig. 2Go. It can be seen that class II stimulation had no effect on CD95 expression whereas CD40 stimulation was able to up-regulate CD95, as previously demonstrated in both mouse and human B cells (1, 35). Similar results were obtained with CH12.LX (data not shown). Thus, although CD95 expression can be up-regulated by CD40 ligation, class II stimulation does not induce any change in CD95 expression, and this cannot be how class II engagement reduces Fas-mediated apoptosis.



View larger version (21K):
[in this window]
[in a new window]
  		FIGURE 2. MHC class II ligation and CD95 expression. A20 cells were stimulated with anti-CD40 (1C10), anti-class II (14-4-4S + 17-3-3S), or isotype control Abs (rat IgG or OKT8, respectively) as described in Materials and Methods for 12 h before immunostaining and flow cytometry analysis for CD95 expression. The thin line represents background staining with a biotinylated isotype control mAb. The thick line is the staining of cells stimulated with anti-class II or isotype control mAbs. The dashed line shows CD95 expression following stimulation through CD40. A representative experiment of three is shown.

 
New protein synthesis is not required to block apoptosis

Previous reports have shown that the transcription of antiapoptotic genes can be responsible for resistance to apoptosis. These studies required sequential ligation of first the rescuing receptor and later CD95. The rapidity with which apoptosis is induced through CD95 (Fig. 1Go) and the occurrence of protection even with simultaneous engagement of both receptors in our experiments suggested that any block in apoptosis induction would occur early in the death pathway. This led to the hypothesis that the rescue by MHC class II would be independent of new protein synthesis. This prediction was tested by performing experiments in the presence or absence of CHX, added to the cell cultures for 30 min before the beginning of the experiment. Approximately 90% of new protein synthesis by A20 and normal splenic B cells and 85% of new protein synthesis by CH12.LX was inhibited during the course of the experiments, as measured by the uptake of [3H]leucine (data not shown). Blocking new protein synthesis did not significantly affect the extent of the MHC class II-mediated rescue in either of the two B cell lines (Fig. 3Go, A and B), nor in normal mouse splenic B cells (Fig. 3GoC). These results suggest that signaling per se causes a biochemical change in the cells which provides a block in the apoptosis pathway.

CD95 and FADD remain associated in rescued cells

We next examined where in the apoptosis pathway class II signals were intervening. The first step in CD95-mediated apoptosis is the association of the adapter molecule FADD. To determine whether CD95 and FADD were associated in cells that had been rescued, we immunoprecipitated CD95 from cells stimulated with anti-CD95, with or without a class II rescue signal, to examine CD95-FADD association. Stimulation at both an early time point (10 min) as well as after apoptosis can be detected (90 min) was tested. CD95 and FADD associated in a ligation-dependent manner (Fig. 4Go, lanes 1 and 2), and this association was not disturbed by the additional ligation of MHC class II (lane 5). Fractions (50 or 25%) of duplicate samples treated with either anti-CD95 or anti-CD95 plus anti-MHC class II were analyzed (lanes 3, 4, 6, and 7), and reductions in the amount of CD95 and FADD loaded can clearly be detected. It is not clear why the proportions of the three FADD bands are somewhat different at the two stimulation time points. However, it is clear that the assay was sensitive enough to detect substantial decreases in CD95-FADD association if they were present, but none were seen. Isotype control Ab also did not interfere with the association of CD95 and FADD. Samples from cells stimulated with MHC class II Abs alone did not show any CD95 or FADD when precipitated with protein G (data not shown).



View larger version (77K):
[in this window]
[in a new window]
  		FIGURE 4. CD95- FADD association was unaffected by MHC class II ligation. CH12.LX cells were stimulated as indicated for 10 or 90 min. To show representative reductions in protein, lanes 3 and 6 contain 50% of the sample amount loaded in lanes 2, 5, and 8; lanes 4 and 7 contain 25%. Anti-CD95-immunoprecipitated proteins were separated by SDS-PAGE and Western blots performed for CD95 and associated FADD. The experiment shown is representative of five independent experiments. Similar results were seen with A20 cells (data not shown).

 
Caspase 8 activity is reduced by rescue signals

The association of CD95 and FADD leads to the rapid recruitment and activation of caspase 8, so we tested whether or not CD95-induced caspase 8 activity was affected by MHC class II signaling. Caspase 8 activity was measured using a fluorometric assay that determines the ability of cell lysates to cleave the caspase 8-specific substrate IETD-AMC. As expected, caspase 8 activity was low in unstimulated cells; this level of activity was arbitrarily assigned a value of 1. The remaining conditions are expressed as fold values over the unstimulated sample. Upon activation of the death pathway by ligation of CD95, caspase 8 activity is increased between 20- and 25-fold in B cell lines and 3- to 4-fold in normal splenic B cells (Fig. 5Go). B cells also treated with anti-class II mAbs contained substantially reduced amounts of caspase 8 activity, whereas the isotype control Ab did not decrease the activity of caspase 8. This pattern is visible in both A20 and CH12.LX (Fig. 5Go, A and B), as well as normal B cells (Fig. 5GoC). These data show that class II-mediated rescue from Fas-induced apoptosis corresponds to a reduction in caspase 8 activation.



View larger version (14K):
[in this window]
[in a new window]
  		FIGURE 5. Class II ligation inhibits CD95-induced caspase 8 activity. A20 (A), CH12.LX (B), or normal splenic B cells (C) were stimulated for 2 h before lysis. Stimuli included BCM only ({square}), anti-Fas mAb ({blacksquare}), anti-Fas + anti-class II mAbs (), and anti-Fas + isotype control mAbs (). Caspase 8 activity in cell lysates was determined by fluorometry using the substrate IETD-AMC, as described in Materials and Methods. Results from unstimulated cells were set at 1 and the remaining treatments are expressed as fold increase above the unstimulated sample. Average readings for the untreated samples were: A20, 0.062; CH12.LX, 0.092; splenic B cells, 1.22. Results are the mean values ± SE of three experiments.

 
Caspase 8 cleavage is blocked by class II-mediated signals

The loss of caspase 8 activity could result from a failure to cleave the zymogen into the active form or from an inhibition of the activated caspase. Caspase 8 cleavage can be observed by Western blotting for the loss of the zymogen or proprotein, or the appearance of the p20 cleavage product, as shown in our previous study (4). Fig. 6Go illustrates that lysates from unstimulated cells showed a modest amount of the p20 cleavage product (lane 1), which increased markedly following Fas ligation (lane 2). Simultaneous ligation of class II MHC effectively blocked the appearance of increased amounts of p20 (lane 3), whereas the isotype control mAbs did not block Fas-mediated caspase 8 cleavage (lane 4). The blot shown in the upper panel was stripped and reprobed for actin (lower panel) to demonstrate similar amounts of protein loaded in each lane.



View larger version (33K):
[in this window]
[in a new window]
  		FIGURE 6. Inhibition of caspase 8 cleavage by class II-mediated signals. A20 cells were stimulated for 3 h and lysates were prepared. Samples were separated by SDS-PAGE and Western blots probed with an Ab specific for the p20 cleavage product of caspase 8 (see Antibodies in Materials and Methods). Cell treatments were as follows. Lane 1, Isotype control Ab for anti-CD95, lane 2, anti-CD95 mAb, lane 3, anti-CD95 + anti-class II mAb, and lane 4, anti-CD95 + isotype control Ab. Stimulating Abs were as in Fig. 1Go. Molecular weights are indicated at the right. The position of the cleaved p20 form of caspase 8 is indicated to the left. The lower panel shows the same blot used in the upper panel, stripped and reprobed for actin to show equal protein loading of the lanes. The band above actin in lane 3 is the H chain (HC) of the mouse anti-class II-stimulating mAb detected by the secondary Ab used to detect anti-actin on the blots. Data presented are representative of three similar experiments; similar findings were seen using CH12.LX cells and a sheep anti-caspase 8 Ab prepared for us by Elimra Biologicals (Iowa City, IA) (data not shown).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A substantial number of previous studies, including several from our laboratory, have demonstrated that ligation of MHC class II molecules can provide activating signals to both mouse (24, 25, 26, 27, 32) and human (36, 37, 38, 39) B lymphocytes. Such signals, although not by themselves crucial for normal B cell activation, can serve to promote the activation of B cells by cognate interactions with T lymphocytes. This is in contrast to many other T cell-dependent B cell activation receptors (e.g., lymphokine receptors, adhesion molecules, and members of the TNF-R family such as CD40) that can be engaged by any activated T cell, regardless of specificity. The ability of class II signals to synergize with BCR (25, 32, 38), lymphokine receptor (40, 41), and CD40 signals (27, 42, 43) is consistent with this idea. This may be of particular importance for selection of B cells to survive in the germinal center. The anatomical location in which B cells are presumably eliminated is the T cell-rich periarteriolar sheath, because this is where autoreactive B cells, including those in CD95-deficient mice, accumulate before elimination (44). Thus, the survival of B cells depends on productive B cell-T cell interactions.

MHC molecules present antigenic peptides to T cells and serve as a ligand for the TCR, which transduces signals to the T cell. This interaction between B and T cells leads to delivery of both positive and negative signals. For example, activated T cells express Fas ligand on their surface membranes and B cells that receive signals though the CD40 molecule are induced to express Fas/CD95. Thus, a productive B and T cell interaction juxtaposes a death receptor and its ligand. We suggest that the B cell receives antiapoptotic signals sufficient to inhibit induction of apoptosis during appropriate cognate interactions. The data presented here suggest that MHC class II participates in these signals. The signals would no doubt include appropriate ligation of the BCR, CD40, IL-4, and MHC class II receptors, all of which provide antiapoptotic signals (3, 4, 6). Clearly maximal ligation of MHC class II is unlikely to occur in vivo. Thus, we propose that full protection would require the concerted effects of multiple antiapoptotic signals from different receptors to successfully block death induction.

Although, as mentioned above, many previous studies have characterized class II as an activating receptor on B cells, several previous reports have also suggested that MHC class II ligation alone induces apoptosis. We have addressed this question in normal mouse splenic B cells and B cell lines but found no decrease in viability resulting from MHC class II ligation. What could account for differences seen between various studies? The majority of studies finding class II-mediated apoptosis used human B cells; therefore, species differences could be a factor. However, another complication is that in many of the studies of human B cells, B cell lines transformed by EBV were used. As it is now known that proteins produced by EBV in B cells can constitutively signal to the infected cells (45), this complicates studies of B cell signaling in EBV-transformed cells. Indeed, in a study using both an EBV-transformed B cell line and small, dense splenic B cells, only the former cells were sensitive to death mediated via class II molecules (46). In another study of human B cell lines, only three of six of the non-EBV-transformed cell lines showed class II-enhanced sensitivity to apoptosis (34). Thus, it is important that our findings in two mature B cell lines (CH12.LX and A20) were confirmed by results using normal splenic B cells, since results between different cell lines may differ. One previous study that also used normal mouse splenic B cells showed that anti-class II treatment increased by 1.6-fold the proportion of cells that were permeable to ethidium bromide (47). This was interpreted as indicating that class II ligation induced apoptosis. There was no DNA laddering, a hallmark of apoptosis, in the untreated cells. However, ~40% were judged apoptotic by their ethidium bromide uptake (47). Thus, these results are difficult to directly compare with the findings of the present study, which used a widely accepted method to identify apoptotic cells.

Previously reported pathways for regulating CD95-mediated apoptosis in B cells are dependent upon the transcription of new proteins. Both caspase 8 inhibitory protein, a specific inhibitor of caspase 8, and Fas apoptosis inhibitory molecule, a protein that inhibits CD95-mediated apoptosis by an unknown mechanism, are up-regulated in B cells following BCR stimulation (10, 48). BCR ligation is also reported to increase expression of the antiapoptotic protein bcl-xL (7). CD40 ligation also induces the expression of antiapoptotic bcl family proteins, which results in resistance to CD95-induced apoptosis (49). Additional mechanisms for the inhibition of apoptosis have been described. These include the expression of TNF-R-associated factors, inhibitor of apoptosis proteins which are direct inhibitors of active caspases, and activation of NF-{kappa}B (50, 51).

Our data reveal an additional pathway for blocking CD95-mediated death. Because apoptosis activated by CD95 occurs quite rapidly and is initiated by the activation of caspase 8, it may not be possible for cells to up-regulate antiapoptotic proteins quickly enough to block caspase 8-mediated damage. Our results indicate that multiple pathways may emanate from a given receptor that affect the sensitivity of a cell to apoptosis induction, suggesting a role for posttranslational modification of proteins involved in CD95-mediated apoptosis. This is reminiscent of the antiapoptotic pathways activated by growth factors such as IL-3, which induces the phosphorylation of Bad and inhibition of apoptosis in response to growth factor withdrawal (52). Caspase 9 contains a regulatory serine, which when phosphorylated inhibits enzyme activity (53). The possibility of modification of caspase 8 or FADD phosphorylation by B cell-activating signals is currently under investigation. We have performed experiments with various inhibitors of protein and lipid kinases as well as inhibitors of cAMP production but have found no specific inhibition of MHC class II-mediated rescue (data not shown). The inability to inhibit rescue is probably due to the numerous classes of kinases and the lack of precise information as to which kinases are used by MHC class II for signal transduction. An alternative explanation is that ligation of antiapoptotic receptors may change or perturb the structure of the plasma membrane in such a way as to inhibit functioning of CD95 as a death receptor. This is a possibility in light of the recent report that MHC class II can translocate to cholesterol-rich lipid microdomains (54). CD95 is a member of the TNF-R superfamily, as is CD40, which translocates to rafts upon receptor cross-linking (55). This reorganization of the plasma membrane may dislocate CD95 from a critical signaling niche and thus block apoptosis induction. Alternatively, this type of rearrangement may juxtapose MHC class II and CD95 and allow direct cross-talk between signaling pathways used by each receptor, a possibility we are exploring.

It is especially important to understand the molecular mechanisms by which cells regulate apoptosis induction for several reasons. Cancerous transformation often includes resistance to apoptosis induction, and pathogenic organisms have evolved mechanisms to silence apoptosis induction that occurs in response to infection. Additionally, CD95 is important in the elimination of autoreactive lymphocytes. Thus, understanding the normal mechanisms that cells use to regulate apoptosis can provide insight for the design of therapies to combat both cancer and infection.


   Acknowledgments
 
We thank Luis Ramirez for excellent technical assistance and Dr. Astar Winoto for providing Ab to FADD.


   Footnotes
 
1 This work was supported by grants from the National Institutes of Health (AI28847 and CA66570) and the Veterans Affairs (Merit Review 383 to G.A.B.). Core support was provided by National Institutes of Health Grant DK25295 to the University of Iowa Diabetes and Endocrinology Research Center. Back

2 Address correspondence and reprint requests to Dr. Gail A. Bishop, Department of Microbiology, University of Iowa, 3-570 Bowen Science Building, Iowa City, IA 52242. Back

3 Abbreviations used in this paper: BCR, B cell Ag receptor; BCM, B cell medium; CHX, cycloheximide; FADD, Fas-associated death domain-containing protein; AMC, 7-amino-4-trifluoromethyl coumarin. Back

Received for publication August 24, 2000. Accepted for publication March 8, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Schattner, E. J., K. B. Eldon, D.-H. Yoo, J. Tumang, P. H. Krammer, M. K. Crow, S. M. Friedman. 1995. CD40 ligation induces Fas expression on human B lymphocytes and facilitates apoptosis through the Fas pathway. J. Exp. Med. 182:1557.[Abstract/Free Full Text]
  2. 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]
  3. 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 CD95-ligands modulated by the B cell antigen receptor. Cell 87:319.[Medline]
  4. Catlett, I. M., G. A. Bishop. 1999. Cutting edge: a novel mechanism for rescue of B cells from CD95/Fas-mediated apoptosis. J. Immunol. 163:2378.[Abstract/Free Full Text]
  5. Cleary, A. M., S. M. Fortune, M. J. Yellin, L. Chess, S. Lederman. 1995. Opposing roles of CD95 (Fas/APO-1) and CD40 in the death and rescue of human low density tonsillar B cells. J. Immunol. 155:3329.[Abstract]
  6. 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]
  7. Koizumi, T., J. Wang, Y. Suzuki, K. Masuda, T. Watanabe. 1996. Regulation of bcl-xL expression and Fas susceptibility in mouse B cells by CD40 ligation, surface IgM crosslinking and IL-4. Mol. Immunol. 33:1247.[Medline]
  8. Schneider, T. J., D. Grillot, L. C. Foote, G. E. Núñez, T. L. Rothstein. 1997. Bcl-x protects primary B cells against Fas-mediated apoptosis. J. Immunol. 159:4834.[Abstract]
  9. Irmler, M., M. Thome, M. Hahne, P. Schneider, K. Hofmann, V. Steiner, J. Bodmer, M. Schröter, K. Burns, C. Mattmann, et al 1997. Inhibition of death receptor signals by cellular FLIP. Nature 387:190.
  10. 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]
  11. Sarma, V., Z. Lin, L. Clark, B. M. Rust, M. Tewari, R. J. Noelle, V. M. Dixit. 1995. Activation of the B-cell surface receptor CD40 induces A20, a novel zinc finger protein that inhibits apoptosis. J. Biol. Chem. 270:12343.[Abstract/Free Full Text]
  12. Chinnaiyan, A. M., C. G. Tepper, M. F. Seldin, K. O’Rourke, F. C. Kischkel, S. Hellbardt, P. H. Krammer, M. E. Peter, V. M. Dixit. 1996. FADD/MORT1 is a common mediator of CD95 (Fas/APO-1) and TNFR-induced apoptosis. J. Biol. Chem. 271:4961.[Abstract/Free Full Text]
  13. Zhang, J., A. Winoto. 1996. A mouse Fas-associated protein with homology to the human FADD protein is essential for Fas-induced apoptosis. Mol. Cell. Biol. 16:2756.[Abstract]
  14. Muzio, M., A. M. Chinnaiyan, F. C. Kischkel, K. O’Rourke, A. Shevchenko, J. Ni, C. Scaffidi, J. D. Bretz, M. Zhang, R. Gentz, et al 1996. FLICE, a novel FADD-homologous ICE-CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex. Cell 85:817.[Medline]
  15. Imai, Y., T. Kimura, A. Murakami, N. Yajima, K. Sakamaki, S. Yonehara. 1999. The CED-4-homologous protein FLASH is involved in Fas-mediated activation of caspase-8 during apoptosis. Nature 398:777.[Medline]
  16. Zhang, J., D. Cado, A. Chen, N. H. Kabra, A. Wintot. 1998. Fas-mediated apoptosis and activation-induced T-cell proliferation are defective in mice lacking FADD/Mort1. Nature 392:296.[Medline]
  17. Varfolomeev, E. E., M. Schuchmann, V. Luria, N. Chiannikulchai, J. S. Beckmann, I. L. Mett, D. Rebrikov, V. M. Brodianski, O. C. Kemper, O. Kollet, et al 1998. Targeted disruption of the mouse caspase 8 gene ablates cell death induction by the TNF receptors, Fas/Apo1, and DR3 and is lethal prenatally. Immunity 9:267.[Medline]
  18. Thornberry, N. A., T. A. Rano, E. P. Peterson, D. M. Rasper, T. Timkey, M. Garcia-Calvo, V. M. Houtzager, P. A. Nordstrom, S. Roy, J. P. Vaillancourt, et al 1997. A combinatorial approach defines specificities of members of the caspase family and granzyme B: functional relationships established for key mediators of apoptosis. J. Biol. Chem. 272:17907.[Abstract/Free Full Text]
  19. Medema, J. P., C. Scaffidi, F. C. Kischkel, A. Shevchenko, M. Mann, P. H. Krammer, M. E. Peter. 1997. FLICE is activated by association with the DISC. EMBO J. 16:2794.[Medline]
  20. Hirata, H., A. Takahashi, S. Kobayashi, S. Yonehara, H. Sawai, T. Okazaki, K. Yamamoto, M. Sasada. 1998. Caspases are activated in a branched protease cascade and control distinct downstream processes in Fas-induced apoptosis. J. Exp. Med. 187:587.[Abstract/Free Full Text]
  21. Memon, S. A., J. Hou, M. B. Boreno, C. M. Zacharchuk. 1998. Apoptosis induced by a chimeric Fas/FLICE receptor: lack of requirement for Fas or FADD-binding proteins. J. Immunol. 160:2046.[Abstract/Free Full Text]
  22. Mourad, W., R. S. Geha, T. Chatila. 1990. Engagement of MHC class II molecules induces sustained, LFA-1-dependent cell adhesion. J. Exp. Med. 172:1513.[Abstract/Free Full Text]
  23. Altomonte, M., C. Pucillo, G. Damante, M. Maio. 1993. Cross-linking of HLA class II antigens modulates the release of TNF-{alpha} by the EBV-B lymphoblastoid cell line JY. J. Immunol. 151:5115.[Abstract]
  24. Bishop, G. A., G. Haughton. 1986. Induced differentiation of a transformed clone of Ly-1+ B cells by clonal T cells and antigen. Proc. Natl. Acad. Sci. USA 83:7410.[Abstract/Free Full Text]
  25. Baluyut, A. R., B. Subbarao. 1988. The synergistic effects of anti-IgM and monoclonal anti-Ia antibodies in induction of murine B lymphocyte activation. J. Mol. Cell. Immunol. 4:45.[Medline]
  26. Cambier, J. C., K. R. Lehmann. 1989. Ia-mediated signal transduction leads to proliferation of primed B lymphocytes. J. Exp. Med. 170:877.[Abstract/Free Full Text]
  27. Bishop, G. A., W. D. Warren, M. T. Berton. 1995. Signaling via MHC class II molecules and antigen receptors enhances the B cell response to gp39/CD40 ligand. Eur. J. Immunol. 25:1230.[Medline]
  28. Cambier, J. C., D. C. Morrison, M. M. Chien, K. R. Lehmann. 1991. Modeling of T cell contact-dependent B cell activation. IL-4 and antigen receptor ligation primes quiescent B cells to mobilize calcium in response to Ia cross-linking. J. Immunol. 146:2075.[Abstract]
  29. Lane, P. J. L., F. M. McConnell, G. L. Schieven, E. A. Clark, J. A. Ledbetter. 1990. The role of class II molecules in human B cell activation: association with phosphatidyl inositol turnover, protein tyrosine phosphorylation, and proliferation. J. Immunol. 144:3684.[Abstract]
  30. Cambier, J. C., M. K. Newell, L. B. Justement, J. C. McGuire, K. L. Leach, Z. Z. Chen. 1987. Ia binding ligands and cAMP stimulate nuclear translocation of PKC in B lymphocytes. Nature 327:629.[Medline]
  31. Bishop, G. A.. 1991. Requirements of class II-mediated B cell differentiation for class II cross-linking and cAMP. J. Immunol. 147:1107.[Abstract]
  32. Bishop, G. A., L. M. Ramirez, T. J. Waldschmidt. 1994. Differential responses to Ig and class II-mediated signals in splenic B cell subsets from normal and autoimmune mice. Int. Immunol. 6:1049.[Abstract/Free Full Text]
  33. Heath, A. W., W. W. Wu, M. C. Howard. 1994. Monoclonal Abs to murine CD40 define two distinct functional epitopes. Eur. J. Immunol. 24:1828.[Medline]
  34. Yoshino, T., L. Cao, R. Nishiuchi, Y. Matsuo, I. Yamadori, E. Kondo, N. Teramoto, K. Hayashi, K. Takahashi, N. Kamikawaji, T. Akagi. 1995. Ligation of HLA class II molecules promotes sensitivity to CD95-mediated apoptosis. Eur. J. Immunol. 25:2190.[Medline]
  35. Hostager, B. S., Y. Hsing, D. E. Harms, G. A. Bishop. 1996. Different CD40-mediated signaling events require distinct CD40 structural features. J. Immunol. 157:1047.[Abstract]
  36. Palacios, R., O. Maza-Martinez, K. Guy. 1983. Monoclonal antibodies against HLA-DR antigens replace T helper cells in activation of B lymphocytes. Proc. Natl. Acad. Sci. USA 80:3456.[Abstract/Free Full Text]
  37. Brick-Ghannam, C., F. L. Huang, N. Temime, D. Charron. 1991. PKC activation via human leukocyte antigen class II molecules. J. Biol. Chem. 266:24169.[Abstract/Free Full Text]
  38. Mooney, N., D. Van Alewyk, C. Brick-Ghannan, D. Charron. 1991. HLA class II antigen-mediated induction of a proliferative response to anti-IgM in human B lymphocytes. Int. J. Cancer 6:30.
  39. Kansas, G. S., J. C. Cambier, T. F. Tedder. 1992. CD4 binding to MHC class II antigens induced LFA-1-dependent and independent homotypic adhesion of B lymphocytes. Eur. J. Immunol. 22:147.[Medline]
  40. Stein, S. H., R. P. Phipps. 1992. Anti-class II antibodies potentiate IgG2a production by LPS-stimulated B lymphocytes treated with PGE2 and IFN-{gamma}. J. Immunol. 148:3943.[Abstract]
  41. Kubota, E., D. T. McKenzie, R. W. Dutton, S. L. Swain. 1991. Role of T cells in the B-cell response: glutaraldehyde-fixed T-helper hybridoma cells synergize with the lymphokine IL-4 to induce B-cell activation and proliferation. Immunology 72:40.[Medline]
  42. Mehindate, K., R. Al-Daccak, F. Damdoumi, W. Mourad. 1996. Synergistic effect between CD40 and class II signals overcomes the requirement for class II dimerization in superantigen-induced cytokine gene expression. Eur. J. Immunol. 26:2075.[Medline]
  43. Poudrier, J., T. Owens. 1994. CD54/ICAM-1 and MHC class II signaling induces B cells to express IL-2 receptors and complements help provided through CD40 ligation. J. Exp. Med. 179:1417.[Abstract/Free Full Text]
  44. Jacobson, B. A., D. J. Panka, K. A. Nguyen, J. Erikson, A. K. Abbas, A. Marshak-Rothstein. 1995. Anatomy of autoantibody production: dominant localization of antibody-producing cells to T cell zones in Fas-deficient mice. Immunity 3:509.[Medline]
  45. Thorley-Lawson, D. A., E. M. Miyashita, G. Khan. 1996. EBV and the B cell: that’s all it takes. Trends Microbiol. 4:204.[Medline]
  46. Truman, J.-P., M. L. Ericson, C. J. M. Choqueux-Seabold, D. J. Charron, N. A. Mooney. 1994. Lymphocyte programmed cell death is mediated via HLA class II DR. Int. Immunol. 6:887.[Abstract/Free Full Text]
  47. Newell, M. K., J. VanderWall, K. S. Beard, J. H. Freed. 1993. Ligation of MHC class II molecules mediates apoptotic cell death in resting B lymphocytes. Proc. Natl. Acad. Sci. USA 90:10459.[Abstract/Free Full Text]
  48. 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]
  49. 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]
  50. Wang, C., M. W. Mayo, R. G. Korneluk, D. V. Goeddel, A. S. Baldwin. 1998. NF-{kappa}B antiapoptosis: induction of TRAF1 and TRAF2 and cIAP1 and cIAP2 to suppress caspase-8 activation. Science 281:1680.[Abstract/Free Full Text]
  51. Deverauz, Q. L., R. Takahashi, G. S. Salvesen, J. C. Reed. 1997. X-linked IAP is a direct inhibitor of cell-death proteases. Nature 388:300.[Medline]
  52. del Peso, L., M. Gonzalez-Garcia, C. Page, R. Herrera, G. Nuñez. 1997. IL-3-induced phosphorylation of Bad through the protein kinase Akt. Science 278:687.[Abstract/Free Full Text]
  53. Cardone, M. H., N. Roy, H. R. Stennicke, G. S. Salvesen, T. F. Franke, E. Stanbridge, S. Frisch, J. C. Reed. 1998. Regulation of cell death protease caspase-9 by phosphorylation. Science 282:1318.[Abstract/Free Full Text]
  54. Huby, R. D., R. J. Dearman, I. Kimber. 1999. Intracellular phosphotyrosine induction by MHC class II requires co-aggregation with membrane rafts. J. Biol. Chem. 274:22591.[Abstract/Free Full Text]
  55. Hostager, B. S., I. M. Catlett, G. A. Bishop. 2000. Recruitment of CD40, TRAF2 and TRAF3 to membrane microdomains during CD40 signaling. J. Biol. Chem. 275:15392.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Int ImmunolHome page
M. A. Kriegel, S. Adam-Klages, C. Gabler, N. Blank, M. Schiller, C. Scheidig, J. R. Kalden, and H.-M. Lorenz
Anti-HLA-DR-triggered monocytes mediate in vitro T cell anergy
Int. Immunol., April 1, 2008; 20(4): 601 - 613.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
G. Chattopadhyay, A. Q. Khan, G. Sen, J. Colino, W. duBois, A. Rubtsov, R. M. Torres, M. Potter, and C. M. Snapper
Transgenic Expression of Bcl-xL or Bcl-2 by Murine B Cells Enhances the In Vivo Antipolysaccharide, but Not Antiprotein, Response to Intact Streptococcus pneumoniae
J. Immunol., December 1, 2007; 179(11): 7523 - 7534.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
C. Stoicov, X. Cai, H. Li, K. Klucevsek, J. Carlson, R. Saffari, and J. Houghton
Major Histocompatibility Complex Class II Inhibits Fas Antigen-Mediated Gastric Mucosal Cell Apoptosis through Actin-Dependent Inhibition of Receptor Aggregation
Infect. Immun., October 1, 2005; 73(10): 6311 - 6321.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
T. O. Nashar and J. R. Drake
The Pathway of Antigen Uptake and Processing Dictates MHC Class II-Mediated B Cell Survival and Activation
J. Immunol., February 1, 2005; 174(3): 1306 - 1316.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
B. S. Hostager, S. A. Haxhinasto, S. L. Rowland, and G. A. Bishop
Tumor Necrosis Factor Receptor-associated Factor 2 (TRAF2)-deficient B Lymphocytes Reveal Novel Roles for TRAF2 in CD40 Signaling
J. Biol. Chem., November 14, 2003; 278(46): 45382 - 45390.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
B.-C. Yang, H.-K. Lin, W.-S. Hor, J.-Y. Hwang, Y.-P. Lin, M.-Y. Liu, and Y.-J. Wang
Mediation of Enhanced Transcription of the IL-10 Gene in T Cells, Upon Contact with Human Glioma Cells, by Fas Signaling Through a Protein Kinase A-Independent Pathway
J. Immunol., October 15, 2003; 171(8): 3947 - 3954.
[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
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Catlett, I. M.
Right arrow Articles by Bishop, G. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Catlett, I. M.
Right arrow Articles by Bishop, G. A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS