1 INSERM U396, Immunogenetique Humaine, Institut Biomedical des Cordeliers, 75006 Paris, France 2 UPRESEA 2233 dHématologie et de la Biologie des cellules sanguines, CHRU de Rennes, Rennes, France 3 MRC Centre for Immune Regulation, University of Birmingham Medical School, Birmingham, UK
The first two authors contributed equally to this work
Correspondence to: N. Mooney; E-mail: nuala.mooney@bhdc.jussieu.fr
Transmitting editor: T. Sasasuki
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Abstract |
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Keywords: HLA class II signals, human dendritic cells, protein kinase C, programmed cell death
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Introduction |
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In addition to their role in antigen presentation, signal transduction via MHC class II molecules has been widely documented and leads to diverse consequences for the MHC class II-expressing cell (3). Recent studies in B lymphocytes and in monocytes have revealed the implication of Syk kinase in the production of IgM (4), and of both ERK and p38 MAP kinases in the production of the pro-inflammatory cytokine IL-1ß (5). Tyrosine phosphorylation of the BCR-associated proteins CD79 and ß via MHC class II engagement was revealed in murine B lymphocytes (6). Moreover, a role for specific microdomains of the plasma membrane has been demonstrated in both signal transduction and in antigen presentation via MHC class II antigens (79). Both the signaling pathways and the consequences of signaling via MHC class II antigens vary depending on the MHC class II-expressing cell and the stimulating ligand. A clear demonstration of the importance of the form of the stimulating ligand is provided by the study of Tabata et al. (4). Whereas fixed HLA class II antibodies induced IgM production, neither apoptosis nor B cell proliferation was induced in the presence of soluble HLA-DR mAb. A consequence of HLA-DR-mediated signaling is apoptosis of mature B lymphocytes which proceeds by an apparently caspase-independent pathway despite the observation of typical membrane and nuclear features of apoptosis (10). We have reported that whereas immature DC were relatively resistant to HLA-DR-mediated apoptosis, mature DC were susceptible to apoptosis and acquisition of susceptibility corresponded to their acquisition of markers defining mature APC (11). Since tyrosine protein kinase and PKC activation are early features of HLA-DR-mediated signals in various APC (6,12), we compared their activation via HLA-DR in both immature and mature DC.
Activation of the PKC family of serine/threonine kinases is implicated in many cellular responses including gene expression, cytoskeletal mobility, proliferation, differentiation and apoptosis (13). HLA class II signaling has diverse effects on PKC including transcriptional regulation and enzyme activation (14). Translocation of PKC- and -ßII (belonging to the classical group of PKC isoenzymes) via HLA-DR has been described (15,16). PKC-
has been described as a anti-apoptotic isoenzyme (17), whereas PKC-
has been described as a pro-apoptotic isoenzyme (18). PKC-
is a member of the novel group of PKC isoenzymes and does not therefore require calcium for activation. The role of PKC-
in apoptosis is at least 2-fold because overexpression of the cleaved 40-kDa catalytic fragment of PKC-
induced the appearance of apoptotic morphology (18) and it has also been described as a substrate for cleavage by caspase-3 (19). Phorbol ester stimulation led to mitochondrial translocation of PKC-
and apoptosis via a cytochrome c-release pathway (20). There is also evidence that PKC-
activation enhances caspase-3 activation (21). A pharmacological inhibitor which is selective for PKC-
has been described (22).
Unlike many hematopoietic cells, mature DC do not have recourse to CD95 mediated apoptosis (23). This study examines the mechanism of an apoptotic pathway mediated by the cell surface molecules responsible for the major function of DC, i.e. antigen presentation. As such this pathway to apoptosis could permit auto-regulation of the DC lifespan.
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Methods |
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Safingol is a specific inhibitor of PKC- and Rottlerin is a selective inhibitor of PKC-
(Calbiochem, Merck Eurolab, Limonest, France). Calphostin C was from Calbiochem. Herbimycin A (3.3 µM) inhibits tyrosine kinases (Sigma, St Quentin Fallavier, France) (24). Sodium orthovadanate (1mM) is an inhibitor of protein tyrosine phosphatases (Sigma, St Louis, MO) (25).
Preparation of DC
DC were prepared from healthy donors as described (5) by culturing monocytes with 800 U/ml GM-CSF and 1000 U/ml IL-4. Half of the medium was replaced at day 3, 5 and 7 by fresh medium containing 800 U/ml GM-CSF and 500 U/ml IL-4. At day 7, 100 U/ml TNF- was added to the culture to induce DC maturation which was confirmed by CD83 expression (11).
Localization of PKC- and -
by confocal microscopy
The subcellular localization of PKC isoenzymes was detected by indirect immunostaining and confocal microscopy as previously described (15,16). Cytospins were prepared with 2.5 x 105 cells stimulated with 5 µg/ml of L243 mAb or IgG2a for 20 min. We have previously detected optimal activation and translocation of PKC via HLA-DR after 20 min (14). Cytospins were fixed in cold methanol before staining with PKC isoenzyme-specific antibodies (Santa Cruz Biotechnologies, Santa Cruz, CA). FITC-conjugated secondary mAb (The Binding Site, Birmingham, UK) were used and nuclei were counter-stained with propidium iodide. Fading of fluorescence was retarded with 1,4-diazobicyclo(2,2,2)octane (Dabco; Merck Eurolab). Immunofluorescence was detected by laser scanning confocal microscopy using a MRC 500 confocal microscope (BioRad, Marnes-la Coquette, France).
Subcellular fractionation of DC
DC (107) were either stimulated via HLA-DR for 5 min or left untreated before incubation in hypotonic HB buffer containing 20 mM Tris (pH 7.4), 2 mM MgCl2, 10 mM EGTA, 2 mM EDTA, 2 mM DTT and a cocktail of protease inhibitors (see below). Cells were then snap-frozen in liquid nitrogen for 30 s before heating to 37°C for 2 min and mechanically lysed by homogenization. Lysates were centrifuged at 1000 g for 10 min at 4°C. The pellet (enriched in nuclei) was resuspended in 100 µl of HB buffer containing 1% Triton X-100 and the supernatant was centrifuged at 10,000 g for 10 min at 4°C. The resulting pellet (enriched in mitochondria) was resuspended in 100 µl HB-T and the supernatant was ultracentrifuged at 100,000 g for 1 h at 4°C. The supernatant (cytosolic fraction) was recovered as well as the insoluble cytoskeletal fraction. The quantity of protein in each fraction was determined and equal amounts of protein (20 µg) were migrated in each well; PKC- was detected by immunoblotting.
Detection of tyrosine phosphorylation/dephosphorylation
Tyrosine phosphorylation was detected by immunoblotting with an anti-phosphotyrosine mAb 4G10 (Upstate Biotechnology, Lake Placid, NY). DC (3 x 106 ) were treated with 20 µg/ml of L243 or IgG2a for 2 min, washed and lysed at 4°C in lysis buffer (50 mM Tris, pH 8, 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS, 1 mM Na3VO4, 10 mM NaF, 10 µg/ml aprotinin, leupeptin and pepstatin, and 1 mM PMSF). Post-nuclear cell lysates were separated by SDSPAGE (12%) under reducing conditions and transferred to nitrocellulose (Hybond ECL; Amersham, Paris, France). After blocking with PBS/0.1% Tween and 3% BSA, the membrane was incubated with 1 µg/ml 4G10 for 1 h followed by horseradish peroxidase-conjugated secondary antibody. Tyrosine phosphorylation was revealed by chemiluminescence using ECL reagents (Amersham).
Detection of apoptosis
Annexin V binding. DC were plated in 200 µl of IMDM/10% FCS at a density of 5 x 105 cells/well, in 96-well plates, in the presence of inhibitors or an equivalent volume of inhibitor diluent (DMSO or ethanol). After a 1-h incubation, 5 µg/ml L243 or IgG2a was added and cells were stained with FITC-labeled Annexin V (Boehringer Mannheim, Meylan, France) in order to quantify apoptosis 6 h later. We have previously validated this method of detecting apoptosis in DC (11)Annexin V binding compared favorably with both propidium iodide uptake and detection of DNA strand breaks.
Where indicated, specific apoptosis was calculated as follow: % specific apoptosis = 100 x [(% of Annexin V+ cells in assay) (% of Annexin V+ cells in control)]/[100 (% of Annexin V+ cells in control)].
Determination of mitochondrial membrane potential (m).
m was evaluated by staining cells (106) with 3,3-dihexyloxacarbocyanine iodide (DiOC6) (Molecular Probes, Eugene, OR) at a final concentration of 40 nM (stock solution 1 µM in ethanol) for 15 min at 37°C in the dark after 6 h of stimulation. The fluorescence emitted by cells was analyzed with a FACScan flow cytometer (BD Biosciences, Le Pont-du-Claix, France) using the FL-1 channel. Mature DC were pretreated for 30 min with either Rottlerin or an equivalent volume of DMSO before addition of either L243 (5 µg/ml) or the isotype control.
Immunoblotting of PKC-
Protein (20 µg) from each subcellular fraction was migrated in a 10% SDSPAGE gel and transferred to Hybond PVDF membrane (Amersham). Membranes were blocked overnight at 4°C with 3% non-fat milk in PBS, 0.1% Tween and then probed with anti-PKC- (Santa Cruz Biotechnologies) at a dilution of 1/3000 before incubating with HRP-conjugated donkey anti-rabbit (Amersham) for 1 h at room temperature. The blot was washed and developed using ECL chemiluminescence (Amersham) according to the manufacturers instructions.
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Results |
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PKC- and -
are expressed in both immature and mature DC
Figure 3(ac) shows the distribution of the PKC- isoform in immature DC after stimulation with IgG2a, with L243 or with the phorbol ester phorbol myristate acetate (PMA) respectively. The distribution of PKC-
was predominantly cytosolic and diffuse in IgG2a treated cells with some punctuate staining at the plasma membrane. Stimulation via HLA-DR led to a less diffuse and/or enhanced staining pattern and more continuous plasma membrane staining (Fig. 3b). PMA stimulation led to nuclear localization. In contrast to PKC-
, HLA-DR-mediated stimulation of immature DC did not perturb the cytosolic localization of PKC-
(Fig. 3e) in comparison with isotype control-stimulated cells (Fig. 3d). PMA treatment enhanced PKC-
expression and particularly at the plasma membrane (Fig. 3f).
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Activation and enrichment of PKC- in the subcellular fraction enriched in nuclei
In order to confirm the HLA-DR-driven relocalization of PKC- which was observed by confocal microscopy, a biochemical approach was taken (Fig. 5). Mature DC were separated into fractions enriched for nuclei (Fig. 5, lanes 1 and 2), mitochondria (Fig. 5, lanes 3 and 4) and cytosol proteins (Fig. 5, lanes 5 and 6), and immunoblotting was carried out to detect PKC-
. After stimulation of mature DC via HLA-DR, an enrichment in the full-length PKC-
protein (78 kDa) level was observed in the nuclear fraction. Arbitrary quantification of the bands corresponding to PKC-
was carried out using the NIH Image-based Scion software (Scioncorp, Frederick, MD) and the ratio of the full-length form of PKC-
in L243 (Fig. 5, lanes 2, 4 and 6)- compared to isotype control (Fig. 5, lanes 1, 3 and 5)-stimulated DC was as follows: nuclear fraction 1.68, mitochondrial fraction 1.26 and cytosolic fraction 0.80. The increases detected in the nuclear-enriched and, to a lesser degree, the mitochondria-enriched fractions were therefore paralleled by a decrease in the cytosolic fraction.
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Tyrosine kinase/phosphatase activation is not required for HLA-DR-mediated death of mature DC
Having observed tyrosine phosphorylation via HLA-DR, we tested whether or not this was necessary for apoptosis of mature DC. Herbimycin A inhibits activation of tyrosine kinases (24), but failed to have any inhibitory effect on the induction of HLA-DR-mediated cell death (Fig. 6). L243 induced specific apoptosis in 37.3 ± 16.5%, pre-treatment and incubation with Herbimycin A did not significantly alter this response (32.1 ± 8.9%). Apoptosis of immature DC was not affected by the presence of Herbimycin A (data not shown).
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HLA-DR-mediated apoptosis of mature DC is inhibited by Calphostin C
Having observed relocalization of the PKC- isoform via HLA-DR in mature DC, we first examined the effect of the highly specific PKC inhibitor Calphostin C on HLA-DR-mediated apoptosis. The inhibitory action of Calphostin C is due to its binding of the regulatory domain of PKC. Inhibition of specific apoptosis was observed in a dose-dependent fashion with almost total inhibition at a concentration of 50 nM, which corresponds well with the reported IC50 for PKC (Fig. 7A, background apoptosis did not exceed 12% under all conditions tested).
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When mature DC were examined, Safingol did not change HLA-DR-mediated apoptosis (Fig. 7B, specific apoptosis was determined as: IgG2a and DMSO 20.6 ± 3.3%, IgG2a and Safingol 25.3 ± 3.8%, L243 and Safingol 40.9 ± 11.5%, L243 and DMSO 47.6 ± 10.6%). On the contrary, addition of Rottlerin significantly decreased apoptosis of mature DC induced via HLA-DR without altering non-specific apoptosis. Apoptosis induced via HLA-DR was 47.6 ± 10% and was reduced to 31.1 ± 3% in the presence of Rottlerin (P < 0.05). The difference between the PKC activation via HLA-DR in immature versus mature DC was further shown by the absence of effect of Rottlerin in immature DC (Fig. 7B, L243 and Rottlerin 29 ± 14.7%, L243 and DMSO 21.4 ± 5%).
HLA-DR-mediated depolarization of the mitochondrial membrane is decreased by PKC- inhibition
In order to confirm the inhibition of HLA-DR-mediated apoptosis as a result of inhibition of PKC-, we examined another characteristic of HLA-DR-mediated apoptosis, that of mitochondrial membrane depolarization. The data shown in Table 1 reveal that the number of mature DC undergoing a decrease in their
m 6 h after addition of L243 mAb to mature DC was markedly decreased when the experiment was carried out in the presence of Rottlerin.
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Discussion |
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The initiating step of HLA class II-mediated antigen presentation is specific TCR-mediated engagement of the appropriate HLA class II molecule, engagement of HLA-DR by specific mAb provides a model for studying the signals transmitted via HLA class II in the course of antigen presentation. The L243 mAb was used in this study since it binds in immediate proximity to the peptide binding site of the DR heterodimer (28).
We and others have previously reported sensitivity of mature DC to apoptosis via HLA class II, whereas immature DC are relatively insensitive (11,23 ). We have examined the early signaling events mediated via HLA-DR in mature versus immature DC in order to identify events implicated in the strikingly different apoptotic response of mature versus immature DC to HLA-DR stimulation.
We report that HLA-DR-mediated signals distinguished immature and mature DC since nuclear relocalization of PKC- only occurred in mature DC. Three complementary approaches were taken to study the localization and activation of PKC-
: confocal microscopy, immunoblotting to detect the full-length form and the catalytic fragment, and inhibition of PKC-
activation by both a broad-spectrum PKC and an isoenzyme selective pharmacological inhibitor.
Tyrosine kinase/phosphatase activation was observed in both mature and immature DC, but does not appear to be implicated in the HLA-DR pathway leading to mature DC apoptosis.
HLA class II-mediated signaling in DC has been reported in studies which did not address the question of apoptosis. For example, Kushnir et al. (29) reported HLA-DR-mediated heterotypic aggregation of DC with B lymphocytes. A critical role for PKC-, -ß and -µ isoenzymes (30) has been previously reported in a human myelomonocytic cell line model of DC differentiation. We observed tyrosine phosphorylation/dephosphorylation events in immature and in mature DC, but found no effect of inhibition of tyrosine kinases or phosphatases on HLA-DR-induced apoptosis. HLA-DR-mediated signaling in DC has been previously reported to induce tyrosine kinase activation which was necessary for expression of IL-1ß mRNA (31). Moreover, in B lymphocytes, HLA-DR-mediated tyrosine phosphorylation has been implicated in activation rather than apoptosis (32).
The role of mitogen-activated protein kinase (MAPK) activation in MHC class II-mediated apoptosis has not been explored, although differential activation of ERK and of p38 MAPK has been reported via HLA-DR, -DP and -DQ, and an equilibrium between the activation of various MAPK is important in determining whether a cell survives or undergoes apoptosis (33). B-chronic lymphocytic leukemia cells undergo apoptosis via a CD20-mediated pathway of p38MAPK activation (34), transformed B cell lines undergo p38 dependent BCR-mediated apoptosis (35) and both of these cell types are sensitive to HLA-DR-mediated apoptosis.
Previous studies of signaling motifs, in both the mouse (36) and in the human (16), revealed that tyrosine kinase and PKC activation were mediated by different regions in the MHC class II ß chain. A role for PKC activation in HLA-DR-mediated death was suggested by the inhibition of HLA-DR-mediated apoptosis of B lymphocytes in the presence of broad-spectrum PKC inhibitors (32). The current study in DC reveals a specific role for the PKC- isoenzyme in HLA-DR-mediated apoptosis in mature DC. The importance of PKC-
activation in HLA-DR-mediated apoptosis of mature DC was confirmed by the inhibition noted in the presence of the broad-spectrum PKC inhibitor Calphostin C and particularly in the presence of Rottlerin. Two complementary approaches were taken to determine apoptosisAnnexin V binding, which detects apoptosis at the level of the plasma membrane, and DiOC6 fluorescence, which specifically determines apoptosis at the level of the mitochondria on the basis of
m. A previous study reported inhibition of activation of PKC-
with Rottlerin at a final concentration of 15 µM in monocytes (22), we have used the same conditions to inhibit apoptosis of monocyte-derived DC.
Previous studies in hematopoietic cells have revealed PKC- as a pro-apoptotic isoform, and include spontaneous neutrophil apoptosis (18), Fas-induced apoptosis of T cells (37) and ionizing radiation-induced apoptosis of myeloid leukemia cells (38). At least two nuclear targets of PKC-
have been identifiedDNA-dependent protein kinase (DNA-PK) (39) and lamin B (40). Interaction of DNA-PK with PKC-
leads to inhibition of its DNA repair function (39) and PKC-
phosphorylation of lamin B is required for lamina disassembly.
Engagement of HLA-DR by mAb as ligands is unlikely to mimic the full range of events initiated in the course of the T cell interaction with an APC. Nonetheless, the TCRMHC class II interaction is an indispensable and initiating step in the peptide-specific interaction. In this respect, it is crucial to note that Matsue et al. (41) revealed apoptosis of DC after interaction with an antigen-specific Th cell clone, whereas apoptosis was not observed when DC were cultured with either peptide or with T cells alone.
In summary, activation of PKC- has been demonstrated as a determining step in the apoptosis of different hematopoietic cells (19,37,38), and these data therefore support the notion that HLA-DR-mediated signals in mature DC integrate a common and potent apoptotic pathway. These data could therefore be important for the design of studies of therapeutic uses of mature DC.
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Acknowledgements |
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Abbreviations |
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APCantigen-presenting cell
DCdendritic cell
DiOC63,3-dihexyloxacarbocyanine iodide
DNA-PKDNA-dependent protein kinase
GM-CSFgranulocyte macrophage colony stimulating factor
HRPhorseradish peroxidase
MAPKmitogen-activated protein kinase
PKCprotein kinase C
PMAphorbol myristate acetate
TPKtyrosine protein kinase
TNFtumor necrosis factor
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References |
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