MHC class II-mediated apoptosis in dendritic cells: a role for membrane-associated and mitochondrial signaling pathways
Martin Leverkus1,
Alexander D. McLellan3,
Martina Heldmann1,
Andreas O. Eggert1,
Eva-B. Bröcker1,
Norbert Koch2 and
Eckhart Kämpgen1
1 University of Würzburg Medical School, Department of Dermatology, Josef-Schneider-Strasse 2, 97080 Würzburg, Germany 2 Department for Immunobiology, University of Bonn, 53117 Bonn, Germany 3 Present address: Department of Microbiology, University of Otago, Dunedin, New Zealand
The first two authors contributed equally to this work
Correspondence to: E. Kämpgen; E-mail: kaempgen-e.derma{at}mail.uni-wuerzburg.de
Transmitting editor: T. Hünig
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Abstract
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Cytotoxic elimination of dendritic cells (DC) in lymphoid tissue represents an important pathway of immune regulation. However, the mechanism of DC removal is still controversial since mature DC are insensitive to death receptor-mediated killing and other surface or soluble molecules mediating DC death in vivo have yet to be characterized. Class II ligation is the only known signal that induces rapid cell death in mature DC, thus our studies have now focused on the requirements for this cell death using the advantages of tools available for both the mouse and human systems. Anti-class II mAb could be grouped into (i) mAb that both bound to class II and caused class II-mediated cell death as well as (ii) those that bound to class II, but did not cause apoptosis. mAb binding stable class II dimers as well as those mAb recognizing either the
or ß chains of class II were found in both groups. Whereas class II-mediated death was enhanced by DCDC homotypic interactions, DC clustering itself was insufficient to induce apoptosis. Although DC death could be inhibited by uncoupling actin filament bundling, the inhibition of various proteases, including the caspases, and protein transport mediators failed to inhibit class II-mediated cell death. Neither Bid, poly-ADP-ribose polymerase, caspases-3, -7 and -8 nor FLICE-inhibitory protein were found to be cleaved during class II apoptosis. Lastly, although class II mAb induced a rapid mitochondrial membrane depolarization in DC, cell death was not inhibited by Bcl-2 over-expression in DC. The independence of this form of apoptosis from protein or RNA synthesis, coupled to the rapidity of the mitochondrial depolarization and the lack of protection by Bcl-2, suggests that mature DC express pre-formed pro-apoptotic molecules that are involved in class II-mediated death.
Keywords: apoptosis, caspase, dendritic cell, mitochondria, MHC class II
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Introduction
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Dendritic cells (DC) are a class of antigen-presenting leukocytes found in most tissues in trace amounts that are capable of stimulating primary T cell responses. Immature DC reside in peripheral tissues like the skin, capture antigen and then migrate to the draining lymphoid organs where they may prime both CD4+ as well as CD8+ T cells [for review, see (1)]. During this process, DC mature with a concomitant increase of the expression of co-stimulatory molecules and decreased capacity to further uptake antigen. Thus, effective DC interactions with T cells occur primarily in secondary lymphoid tissues, such as spleen or lymph node, where the migration of lymphocytes and DC throughout areas of high cell density ensures sufficient cellcell contact for immune activation. Close encounters between lymphocytes themselves, as well as between lymphocytes and DC, may lead to apoptosis of these leukocytes (2). The role of apoptosis induction for the elimination of activated lymphocytes has been extensively demonstrated (3); however, knowledge of the fate of DC following activation of cognate T cells is sparse. Thus, revealing the mechanisms regulating DC survival and cell death are crucial for a better understanding of the regulation of immune responses. Recent reports suggested that the signaling mechanisms required for elimination of DC may involve DCT cell as well as DCDC interactions (2,4,5). In addition to the well-characterized receptor-mediated apoptotic signal by death receptors, several other surface molecules are able to induce apoptosis, including CD4, CD45, CD99, CXCR4, MHC class I and BCR. These death-inducing signals differ from the prototypical death receptor-mediated apoptotic signaling pathway with respect to their intracellular enzymatic requirements (611). MHC class II molecules are expressed on the surface of professional antigen-presenting cells (DC, B cells and monocytes) and can be induced by pro-inflammatory cytokines on the surface of many other cell types. The major function of MHC class II is the presentation of peptides derived from exogenous, viral or tumor antigens to CD4+ T lymphocytes. MHC class II molecules are assembled and loaded with antigenic peptides in the endosomes, and subsequently presented on the cell surface as stable heterodimers (12). While the antigen-presenting capabilities of MHC class II have been elucidated in great detail, several studies over the past decade have pointed to an additional role of MHC class II in signal transduction for cells expressing this molecule. Transmission of signals may involve the intracellular domain of the ß chain for some, but not all, activated signaling pathways identified so far (1315). More recently, MHC class II signals have been shown to mediate cell death in B lymphocytes (1618), monocytes (19), and murine and human DC (20,21), but detailed characteristics of MHC class II-mediated apoptosis are missing. The perturbation of the mitochondrion during apoptotic processes, leading to the loss of the inner mitochondrial membrane potential, represents an important point of no return in apoptotic signaling pathways and may lead to concomitant release of proteins from the intermitochondrial membrane space, such as cytochrome c, apoptosis-inducing factor (AIF) and HtrA2 (2225).
We have previously shown that only very immature DC are susceptible to apoptosis mediated via death receptors such as CD95 and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptors, while mature DC are highly resistant to cell death after binding of these ligands (26,27). Thus, the question remains as to which signals may be responsible for the removal of mature DC populations from the lymph node. A potential clue to this problem is the fact that class II ligation of DC provides a strong stimulus for the cell death of mature DC. This, or a related pathway, might be responsible for the rapid demise of antigen-loaded, mature DC in the lymph node. In this report, we have examined signaling pathways involved in MHC class II-mediated apoptosis in human and murine DC, and demonstrate the involvement of membrane-associated and mitochondrial signaling pathways.
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Methods
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Materials
The protease inhibitor z-Val-Ala-Asp-fluoromethyl ketone (ZVAD-fmk) was obtained from Bachem (Heidelberg, Germany). The following mAb were used: mouse anti-human Bcl-2 antibodies (PharMingen, Heidelberg, Germany), anti-poly-ADP-ribose polymerase (PARP) mAb (G. Poirier, CHUL Research Center, Quebec, Canada), anti-CPP32 polyclonal antibodies (D. Nicholson, Merck Frosst, Quebec, Canada), polyclonal rabbit caspase-7 (28), polyclonal rabbit anti-Bid antibodies (provided by Dr X. Wang, Howard Hughes Medical Center, Houston, Texas), FADD-like ICE [(caspase-8) FLICE] mAb (C-15) (29) and cellular FLICE-inhibitory protein (cFLIP) antibodies (NF-6) (30). Recombinant TRAIL, CD95L, CD40L and TRANCE were obtained from Alexis (Gruenberg, Germany). mAb to CD83, CD1a, CD14, CD36, CD64, CD83, CD86 and CD25 were obtained from Dianova (Hamburg, Germany). Rat anti-mouse CD86phycoerythrin was obtained from PharMingen. The mAb to mouse class II molecules are listed in Table 1. Horseradish peroxidase- and FITC-conjugated goat anti-mouse IgG were from PharMingen. Tetramethyl-rhodamine ethyl ester (TMRE) and 3,3'-dihexyloxa carbocyanine iodide [DiOC6(3)] were obtained from Molecular Probes (Eugene, OR).
Preparation of human DC
Human DC were prepared according to Romani et al. (31). Briefly, peripheral blood mononuclear cells were isolated from heparinized buffy coats of healthy adult donors by adherence to plastic cell culture dishes in RPMI 1640 medium supplemented with 0.5% autologous plasma, 2 mM L-glutamine, 50 µg/ml gentamicin and 100 U/ml granulocyte macrophage colony stimulating factor (GM-CSF) for 60 min. The non-adherent cells were removed by gently washing twice with warm PBS. Adherent monocytes were cultured for 7 days in RPMI 1640 medium (Gibco, Eggenstein, Germany) supplemented with 1% autologous plasma, 2 mM L-glutamine, 50 µg/ml gentamicin, 500 U/ml rhIL-4 (Strathmann, Hannover, Germany) and 800 U/ml rhGM-CSF (Leukomax; Sandoz, Basel, Switzerland). For the maturation of DC, day 7 cells were cultured in a cytokine cocktail of IL-1
(500 U/ml), IL-6, TNF-
, IL-1ß (all 1000 U/ml; Strathmann Biotech, Hannover, Germany) and prostaglandin E2 (108 M; Sigma, Deisenhofen, Germany) for another 3 days (32). Early DC progenitor populations were harvested after 2 days of culture as floating vital cells (as determined by Trypan blue exclusion) and incubated overnight with death ligands as described below. Mature DC were defined by morphological (e.g. motile cytoplasmic processes), phenotypical (e.g. expression of CD83, CD25 and MHC at high levels) and functional (allo-T cell stimulatory capacity) characteristics. Jurkat T cells were cultured in RPMI containing 5% FCS. Cell culture supernatants were obtained from the hybridomas L227 (IgG1), L243 (IgG2a), 9.3F10 (IgG1) and CD40 mAb (clone G28.5, IgG1; all from ATCC).
Preparation of murine bone marrow-derived DC (BMDC)
AWY, C57Bl/6 or BALB/c mice were purchased from M & B (Bomholtgard, Denmark) and class II (C57BL/6 Ab/)-deficient mice were obtained from Taconic (New York, NY). Mice expressing the human bcl-2 gene under the control of the vav promoter, which ensures expression of human Bcl-2 in all hematopoietic cells (33), were kindly provided by A. Strasser and J. M. Adams (WEHI, Melbourne Australia). All mice were normally used at 46 weeks of age and were housed in the animal facilities at the University of Würzburg. Media used throughout the study was RPMI 1640 or IMDM supplemented (R10 and I10 respectively) with 10% heat-inactivated FCS (Biowhittaker, Belgium), 50 µM ß-mercaptoethanol, 2 mM glutamine, 100 µg/ml penicillin and 50 µg/ml streptomycin. For DC culture, R10 was supplemented with 5% of culture supernatant from the murine GM-CSF-secreting Ag8653 myeloma line (34). The sources of the mAb used in this study are shown in Table 1. BMDC were generated exactly as previously described (35). Briefly, 24 x 106 bone marrow cells (without red cell lysis or removal of mature cell lineages) were plated in 10 ml of R10 plus GM-CSF, with addition of 10 ml fresh media at day 3, and, by gently discarding 10 ml media, replacing with fresh media at days 6 and 9. BMDC cultures were harvested at days 911 after lipopolysaccharide treatment (200 ng/ml; overnight) as a maturation stimulus. DC were replated at 5 x 105 cells/ml with R10 plus GM-CSF into 24- or 48-well plates (Falcon).
FACScan analysis
For analysis of 
m, 5 x 105 cells were resuspended in supplemented DMEM containing 80 nM DiOC6(3) or 40 nM TMRE and incubated at 37°C for 15 min as described (36). After washing once with ice-cold PBS, cells were immediately analyzed. For all experiments, 105 cells were analyzed by FACScan (Becton Dickinson, San Jose, CA). For the assessment of phenotypic differentiation and maturation of DC, surface expression was essentially performed as described elsewhere (27).
Induction and inhibition of apoptosis in DC
Immature DC (day 7) as well as mature DC (day 10) were seeded at a density of 1 x 105/ml and were incubated with the indicated concentrations of hybridoma L227 or L243 supernatants. Apoptosis was detected 416 h later by AnnexinFITC/propidium iodide (PI) double staining. In some experiments, externalization of phosphatidylserine on the DC membrane was detected by phycoerythrinAnnexin staining (30 min, 4°C; PharMingen). PI (5 µg/ml) was added immediately prior to analysis by FACS. For BMDC, class II-mediated cell death of mature DC was induced by incubating DC cultures overnight with 10% anti-class II mAb culture supernatant and subsequently analyzed by DiOC6(3) or TMRE staining for 15 min at 37°C. The cultures were then harvested, washed in BSA/PBS and stained with CD86phycoerythrin (PharMingen; 50 µl of 2.5 µg/ml) for 10 min on ice. Pellets were then resuspended in 200 µl BSA/PBS. To calculate absolute numbers of remaining viable cells in the DC cultures we utilized a set volume analysis as previously described for cell counting and cytotoxicity assays (37,38). Briefly, following a 5-s equilibration period at the start of each sample, equivalent volumes from each tube were acquired for 20 s using a FACScan. The number of DiOC6(3)+/CD86+/PI events was then expressed as a percent relative to control tubes. For the co-culture experiments, DC were labeled with 2 µM 5-chloromethylfluorescein diacetate (Molecular Probes) in IMDM at 37°C for 10 min and then washed 4 times in warm I10.
Western blot analysis
Total cellular proteins of mature human DC or BMDC were collected as described (39,40) with the exception that Complete protease inhibitor cocktail (Boehringer, Mannheim, Germany) was used. Protein (50 µg) was electrophoresed on SDSPAGE gels and transferred to nitrocellulose membranes. Membranes were blocked and hybridization was performed with antibodies to caspase-8, cellular FLICE-inhibitory protein (cFLIP), caspase-3, PARP, Bid or caspase-7 as described (40). After incubation with appropriate secondary antibodies, bands were visualized using the ECL detection kit (Amersham, Arlington Heights, IL). Blots were subsequently incubated with anti-tubulin mAb (Sigma) to control equal loading of the gel.
Transmission electron microscopy
Mature human DC were either cultured in media only or treated with 10% L227 culture supernatant for 4 h. Cell pellets were fixed in 1.5% glutaraldehyde, post-fixed in 1% osmium tetroxide, dehydrated stepwise in ethanol, quenched in propylene oxide, resin embedded and ultrathin sections cut for transmission electron microscopy.
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Results
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Characteristics of mAb inducing class II cell death in human and murine DC
We recently characterized MHC class II-induced apoptosis in mature murine DC populations (20,26) and have now analyzed this form of MHC class II-mediated cell death in human DC. First, we examined different mAb against HLA-DR and -DQ for their ability to induce apoptosis in monocyte-derived DC. The mAb L243 (anti-HLA-DR) and L227 (anti-HLA-DQ) rapidly induced apoptosis in fully mature DC as determined by phosphatidylserine externalization and transmission electron microscopy (Fig. 1). Morphological changes included membrane blebbing, chromatin condensation, and severe cytoplasmic and organelle disorganization after only 4 h MHC class II cross-linking, confirming a recent report (21). Whereas death receptor-mediated apoptosis plays an important role for killing of immature DC, fully mature human DC are highly resistant to different death receptor-mediated signals (27). We thus were interested to study HLA class II-mediated cell death during different stages of DC differentiation and maturation. Immature DC maintained in GM-CSF and IL-4 for 10 days showed intermediate levels of HLA-DR on the surface and were markedly less sensitive to HLA-DR- or -DQ-mediated cell death (Fig. 2), confirming a recent report (21). These data suggest that the maturation stage of DC as well as the surface expression of MHC class II molecules are important predictors of MHC class II-induced apoptosis in DC. In contrast, isotype-matched mouse IgG did not induce cell death, thereby excluding Fc receptor-mediated effects on this form of cell death. In addition, MHC class II Jurkat or U937 cells were completely resistant to MHC class II-induced apoptosis (data not shown). Death of mature human DC depended on the dose of anti-MHC class II mAb L243 or L227, but did not require additional cross-linking with rabbit anti-mouse IgG (Fig. 1B). Interestingly, the mAb 9.3F10 (recognizing HLA-DR/DQ) did not induce cell death despite similar staining efficiency as determined by FACS (not shown). Because of the availability of a large panel of allo-type-specific mAb in mouse, we extended this analysis to murine DC and screened a panel of 30 pan-class II and allotype-specific mAb directed against murine class II antigens using DC from three strains of mice. As shown in Table 1 and Fig. 3, several additional mAb against class II were identified that induced apoptosis in DC (group 1 in Fig. 3). Killing of DC correlated with staining intensity, although for all mice strains, several anti-class II mAb were identified that labeled DC strongly, but induced little or no apoptosis (group 2 in Fig. 3). The mouse strain specificity of the labeling, the lack of reaction of these mAb on class II/ mice and the pattern of class II molecules precipitated using the group 2 mAb confirmed their specificity for class II molecules (Table 1 and our unpublished data). Interestingly, mAb recognizing only stable class II dimers as well as mAb that bind to the
or ß class II chains were found in both groups. Comparing these data to results obtained in human DC, the mAb L227 and L243 are group 1 mAb, whereas 9.3 F10 is a group 2 mAb.

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Fig. 1. Triggering of MHC class II induces rapid apoptosis in mature DC. (A) Mature human DC were treated with 10% culture supernatant of hybridoma L227 or isotype-matched control antibody for 4 h and subsequently stained with phycoerythrin-conjugated Annexin-V antibody (left panel) or analyzed by transmission electron microscopy. One representative experiment of a total of three independent experiments. (B) Dose-dependent cell death after treatment with increasing concentrations of MHC class II antibody. Mature human DC were treated with increasing concentrations of hybridoma L227 with or without further addition of 5 µg/ml of rabbit anti-mouse IgG. One representative experiment of a total of three independent experiments.
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Fig. 3. Grouping of mAb to murine class II according to cell-surface binding and killing patterns. Each square represents the staining and killing pattern of different anti-class II mAb on BALB/c DC plotted as mean fluorescence intensity (x-axis) versus the percentage killing (y-axis) with each mAb, as calculated from viability loss induced after a 3-h incubation at 37°C. One of two representative experiments.
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Correlation of cluster formation with DC death
MHC class II are the crucial molecules for antigen presentation to CD4+ T lymphocytes. Furthermore, it is well known that MHC class II-mediated signals lead to homotypic aggregation in B cells (41,42) and DC (20). This homotypic adhesion is not an artifact of linking DC by divalent mAb interactions with class II expressed by two DC, since it is inhibited by cytochalasin D (20). When we examined this phenomenon in more detail, we noted that all reagents able to interfere with class II death such as cytochalasin D or okadaic acid (20) also blocked DCDC homotypic adhesion. Moreover, we noted that all anti-MHC class II mAb able to induce cell death induced rapid homotypic aggregation in DC cultures within 1560 min, correlating with loss of viability. Thus, we further examined the role of homotypic interactions of DC for the induction of MHC class II-induced apoptosis. As shown in Fig. 4, there was a strong correlation between the induction of homotypic adhesion with class II apoptosis for the panel of mAb tested. Moreover, the most effective cell death upon anti-class II mAb treatment occurred at high cell densities in both bone marrow and splenic DC (Fig. 5). Anti-class II-treated DC also failed to die when incorporated into collagen gels, where cellcell contact between cells is minimal (data not shown). Similarly, when class II-induced DC homotypic clustering was blocked by repeated resuspension of the cells no DC death was detectable (data not shown). In addition, murine LC present at only 12% in epidermal cell suspensions were resistant to class II-mediated apoptosis unless purified free of contaminating keratinocytes [(20) and data not shown]. To investigate if clustering is the only determinant of cell death in our experimental systems, we compared anti-HLA class II mAb with a monoclonal anti-CD40 mAb (clone G28.5) in human DC. We detected rapid and strong clustering with similar kinetics as compared to anti-class II mAb (Fig. 6); however, without any induction of apoptosis in DC. This clearly demonstrates that clustering per se is not a sufficient stimulus for inducing DC death. To determine if clustering might induce bystander killing or fratricide of DC in anti-class II-treated cultures, we incubated DC from class II-deficient mice with wild-type DC in the presence of the killing 2G9 anti-class II mAb. The different populations were identified by differential labeling in the co-cultures with cell tracker dyes, as described (37). However, class II/ DC failed to die in these cultures, demonstrating that the class II molecules themselves, or molecules closely associated with them, are required to induce apoptosis in DC (Fig. 7). Taken together, our data demonstrate that MHC class II-mediated cell death requires homotypic cell interactions of murine as well as human DC.

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Fig. 4. Correlation of clustering with DC death. For the cluster analysis in (A), DC were plated in wells of a 200-µl flat-bottom 96-well plate. The degree of clustering (y-axis) was scored as the percentage of DC forming clusters after 4 h after gently resuspending DC 3 times with a 200-µl pipette (+ = 2040%, ++ = 4070%, +++ = 70100% of cells in clusters). The x-axis shows the mean fluorescence intensity value of DC stained by each class II mAb. In (B) degree of clustering (y-axis) is plotted against the percent killing (x-axis) induced by the same mAb, as detailed in Fig. 3. One of two independent experiments.
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Fig. 5. Inhibition of class II-mediated cell death by decreasing cell density. BALB/c DC were incubated for 2 h at the indicated cell densities in flat-bottom plates with or without the addition of 10% anti-class II culture supernatant (mAb 2G9). Plotted viabilities shown in (B) are normalized to the level of spontaneous cell death seen in absence of class II triggering. In (A) the percent of cells showing normal levels of mitochondrial membrane potential are plotted as a percent. One of three independent experiments.
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Fig. 7. Lack of bystander killing of class II/ cells. C57BL/6 DC were labeled with CMFDA diacetate and incubated with unlabeled class II/ DC in the absence or presence of anti-class II mAb (2G9; 10%) for 2 h, and then stained with TMRE. The percent of DC showing normal levels of mitochondrial membrane potential after the incubation period is indicated. One of three independent experiments.
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Involvement of Bcl-2-independent mitochondrial signaling pathways in class II-mediated apoptosis
Having established MHC class II-induced apoptosis as effective for the elimination of DC, we next investigated potential intracellular signaling pathways mediating this form of cell death. Mitochondrial signaling pathways have been implicated in many forms of cell death and the loss of the mitochondrial transmembrane potential has been shown to be an important point of no return of cell death (43). We therefore tested the mitochondrial transmembrane potential (MTP) after MHC class II ligation in human monocyte-derived DC. Interestingly, a rapid time-dependent decrease of MTP was detected within 1560 min following addition of HLA-DR or -DQ mAb (Fig. 8). Importantly, loss of MTP preceded the staining with PI, suggesting that the activation of mitochondrial signaling pathways precedes ultimate cell death. Since Bcl-2 family members have been shown to inhibit mitochondrial signaling pathways of apoptosis (44), we utilized transgenic mice and examined if over-expression of Bcl-2 is able to prevent the rapid MTP loss as well as class II induced cell death. Surprisingly, despite the ability of Bcl-2 to protect lymphocytes against CD95-mediated apoptosis (33), Bcl-2 failed to protect DC from class II-induced apoptosis despite high expression levels of this anti-apoptotic molecule in BMDC (Fig. 9). Although most forms of cell death require the activation of caspases (45), several receptor-mediated forms of cell death proceed independently of this important protease family (611). We therefore analyzed the role of caspases in MHC class II-induced death of DC. Pre-incubation of mature human DC with 40 µM ZVAD-fmk, a well-known cell-permeable irreversible pan-caspase inhibitor, did not protect human DC against the early loss of mitochondrial transmembrane potential or subsequent cell death (Fig. 10). In contrast, TRAIL- or CD95L-mediated apoptosis of immature DC populations could be fully inhibited by this concentration of ZVAD-fmk (not shown). To examine activation of caspases in MHC class II-induced DC apoptosis in more detail, we analyzed cellular lysates of human mature DC 4 h after MHC class II triggering. At this timepoint, >8090% cell death was detected (cf. Fig. 8). However, biochemical analysis of several known initiator (caspase-8) or effector caspases (caspase-3 and -7) by Western blotting did not demonstrate activation of any of these molecules, whereas these proteins were readily cleaved in death receptor-mediated apoptosis of Jurkat cells (Fig. 10B). Within the Bcl-2 family of proteins, the subfamily of BH-3-only proteins, including Bid, have been implicated in different caspase-independent forms of cell death, which bypass Bcl-2 protection of mitochondrial signaling pathways of apoptosis (46). In order to rule out that Bid is cleaved by caspase-independent signaling pathways like granzyme B, we examined Bid cleavage by Western blotting during class II-induced apoptosis (Fig. 10B). However, we found no detectable cleavage of Bid, indicating that class II-induced cell death utilizes Bid-independent signaling pathways not influenced by Bcl-2 for the disruption of mitochondrial function. Since class II killing in murine BMDC is also independent of caspase activation (20), we investigated BMDC to study the involvement of other proteases in the apical and downstream events of class II apoptosis. Surprisingly, a large panel of inhibitors targeting various enzymes including endopeptidases, proteases, matrix metalloproteinases, ceramidases and polymerases failed to significantly inhibit class II-mediated apoptosis (Table 2). In summary these data suggest that MHC class II signals rapidly induce loss of the mitochondrial membrane potential, leading to a fast form of cell death not inhibited by pan-caspase inhibitors or over-expression of Bcl-2. It remains to be shown whether known caspase-independent mitochondrial targets such as AIF, HtrA2 or endonuclease G are involved in downstream events of MHC class II signaling (2325,43,47).

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Fig. 9. Over-expression of Bcl-2 does not protect against class II-mediated cell death. DC were generated either from mice over-expressing the human Bcl-2 gene or wild-type C57Bl/6 DC and tested for their sensitivity to class II killing induced by the 2G9 mAb. The x-axis shows the dilution of class II supernatant and the y-axis the percent of viable DC remaining after 3 h treatment at 37°C. Inlay shows over-expression of human Bcl-2 (upper) or tubulin (lower) in BMDC of transgenic (Bcl-2), but not wild-type (B6), animals used for the experiment. One of three independent experiments.
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Discussion
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The elimination of DC following antigen presentation represents an important regulatory mechanism for the down-regulation of primary immune responses. Several reports have suggested a role for death receptor-mediated signals in the induction of DC apoptosis (4850). In contrast, we and others showed that mature DC are highly resistant to several candidate pathways of apoptosis induction, including death receptor (27,51) and cytotoxic T lymphocyte-induced, granzyme B-mediated apoptosis (52). Thus, additional pathways of DC elimination also involving DCDC interactions may contribute to the termination of antigen presentation, as recently suggested (4,5,53). This paper expands on previous reports from our group and others, demonstrating that MHC class II-mediated cell death might represent a new and important signaling pathway for the elimination of mature DC in the lymph node (20,21). In contrast to CD95 and TRAIL receptor expression, which are unchanged during maturation, class II is up-regulated on the cell surface with a concomitant increase in susceptibility to class II-induced apoptosis, suggesting that surface expression clearly correlates with the sensitivity to class II-mediated cell death. The intracellular mechanisms of cell death following MHC class II cross-linking have not been elucidated in detail. We found that several mAb to HLA-DR and -DQ, as well as newly identified mAb to mouse class II were able to rapidly kill DC. Interestingly, we also identified human and mouse mAb to class II unable to induce DC apoptosis despite similar staining efficiency. Thus, although no particular epitope was identified in either human or mouse class II that triggers cell death, our characterization of two groups of mAb with divergent potential to induce DC apoptosis points to a defined binding requirement for triggering the cell death signal. In addition, our data demonstrate similar signaling pathways of MHC class II-induced apoptosis in different mammals.
Interestingly, class II apoptosis proceeds without a detectable activation of caspases. This independence of caspases was confirmed using cell-permeable inhibitors of caspases like ZVAD-fmk, similar to reports in B cells (18,42). Although we cannot formally exclude that the inhibitors are inefficient for certain known or unknown caspases, our data and others suggest that caspases are not critically involved in MHC class II-induced apoptosis (20,21,26). Furthermore, in line with a previous report in monocytes (19), the involvement of death receptors is highly unlikely based on the fact that (i) pan-caspase inhibitors that readily inhibit death receptor-mediated apoptosis in susceptible human DC populations are unable to block HLA-DR- or -DQ-mediated cell death and (ii) recombinant death ligands like TRAIL or CD95L are unable to kill mature DC populations even at high concentrations of these proteins (27).
Although we demonstrate that a hallmark of class II-mediated cell death is a rapid depolarization of the mitochondria, over-expression of Bcl-2 in DC failed to protect against class II-mediated cell death. Thus, class II apoptosis utilizes Bcl-2-independent pathways for the execution of cell death. However, our data do not exclude that other members of the Bcl-2 family, particularly within the BH3-only subfamily (e.g. Bim or Bmf), may play a distinctive role in signal transduction upon interference with the cytoskeleton and may be involved in class II-induced cell death (54,55). For instance, a recent report demonstrated that loss of Bim leads to complete protection of the phenotype induced by the absence of Bcl-2 (56). Thus, further studies are required to delineate which Bcl-2 family members are expressed in different DC populations.
The physiological relevance of homotypic interaction has not been investigated in detail, but it may be important in the context of DCT cell interactions for augmenting the total DC surface available for T cells. In particular, the qualitative as well as quantitative contact between DC and different T cell populations or homotypic DC interactions may be of fundamental importance for the fate of DC in the lymph node (57). In this regard, cell death of DC may well be influenced by the quantity of DCDC interactions. Our data examining the role of DC clustering suggest that DCDC contact is an essential requirement for this form of apoptosis, although we failed to inhibit class II-induced cell death using a panel of blocking mAb to adhesion molecules expressed on DC (38), including LFA-1, ICAM-1 and -2 and
and ß integrins as well as a wide range of lectins and sugars (Table 3 and data not shown). Moreover, enzyme inhibitors revealed no obvious role for intracellular transport, various proteases or the proteasome complex (Table 2). Similar to our data, Leveille et al. have shown that continuous resuspension of B cell cultures during anti-class II mAb treatment reduces the amount of cell death in B cells (42). However, our data regarding class II apoptosis differ from data obtained using CD95 ligation on T cell clones or hybridomas. In these studies, limiting dilution experiments showed that cell death could still occur on single cells via autocrine CD95LCD95 interactions (58). It is puzzling that DC apoptosis is enhanced by DCDC contact, but this may reflect the requirement for a large area of membrane contact between apoptosis-inducing molecules and their putative ligands expressed on the membranes of the DC. Interestingly, class II/ DC failed to die in co-cultures with class II+/+ cultures (Fig. 7), demonstrating that the class II molecules themselves, or molecules closely associated with them, are required to induce apoptosis in DC. Thus, the signaling mechanism of class II killing may require the class II molecules themselves, as well as molecules closely associated with the class II surface complex.
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Table 3. Inhibition studies for anti-class II mAb-mediated cell death by antibodies to adhesion molecules in BMDC
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Recently, a redistribution and recruitment of HLA-DR to glycolipid enriched membrane microdomains on the surface of Raji cells was detected upon cross-linking of MHC class II (HLA-DR) together with a redistribution of the cytoskeleton. The authors suggested that ligand (or mAb) binding modifies the cytoskeleton at the site of ligand interaction with MHC class II (59). In line with those findings, we and others have observed the complete inhibition of MHC class II-mediated apoptosis following cytochalasin D treatment (20). In addition, the fact that homotypic interaction is necessary, but not sufficient, for efficient MHC class II-induced apoptosis is compatible with this hypothesis. Moreover, in line with our experiments using limiting dilution and repeated resuspension, Tabata et al. demonstrated that immobilization of L243 mAb to the culture plates completely inhibits class II-mediated cell death in B cells (60).
Taken together, our report contributes to the growing body of evidence that mature DC are highly susceptible to class II-mediated apoptosis, thereby representing one of the few mechanisms that may function for the control of DC populations in vivo. Interestingly, in addition to its physiological role to present antigen and interact with T cells, the MHC class II molecule can also be bound by non-professional ligands like bacteria and trigger apoptosis (61). Likewise, bacterial superantigens bind class II and activate monocytes to secrete TNF-
and IL-1ß, and also stimulate B cell homotypic adhesion, thus these toxins may also have the potential to eliminate antigen-presenting cells via class II. Therefore, MHC class II-mediated cell death might also be a mechanism utilized by pathogens to effectively down-regulate immune responses by apoptotic elimination of DC populations.
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Acknowledgements
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The authors would like to thank Drs Ursula Bommhardt, Patrizia Stoitzner, Thomas Hünig, Wolfgang Fischer, Manfred Lutz, Peter Friedl, Martin Sprick and Henning Walczak for assistance with mice, assays, antibodies and cell lines. Bcl-2 transgene mice were generously provided by A. Strasser and J. M. Adams. We thank P. H. Krammer for mAb to caspase-8 (C-15) and cFLIP (NF-6), D. Nicholson for caspase-3 serum, X. Wang for rabbit serum to Bid, and G. M. Cohen for rabbit serum to caspase-7. We are also grateful to Evi Horn, Claudia Kurzmann and Christian Linden for excellent technical assistance. Part of this study was funded by grants of the Sander-Stiftung (2000.092.1) and Deutsche Krebshilfe (10-1951) to M. L., and the German Ministry for Education and Research (12KF-019603 to A.D.M. and E.K., BMBF01KX9820/L and SFB 465 to E.K.). N.K. was supported by SFB 502 and grants from the DFG.
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Abbreviations
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AIFapoptosis-inducing factor
BMDCbone marrow-derived dendritic cell
cFLIPcellular FLICE-inhibitory protein
DiOC6(3)3,3-dihexyloxacarbocyanine iodide
FLICE FADD-like ICE (caspase-8)
MTPmitochondrial transmembrane potential
PARPpoly-ADP-ribose polymerase
PIpropidium iodide
TMREtetramethyl-rhodamine ethyl ester
TNFtumor necrosis factor
TRAILTNF-related apoptosis-inducing ligand
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