Skip Navigation


International Immunology Advance Access originally published online on April 19, 2007
International Immunology 2007 19(6):719-732; doi:10.1093/intimm/dxm034
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
19/6/719    most recent
dxm034v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Majewski, M.
Right arrow Articles by Boes, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Majewski, M.
Right arrow Articles by Boes, M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?


© The Japanese Society for Immunology. 2007. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Protein kinase C delta stimulates antigen presentation by Class II MHC in murine dendritic cells

Michael Majewski1, Tina O. Bose1, Fenna C. M. Sillé1, Annette M. Pollington2, Edda Fiebiger3 and Marianne Boes1,4

1 Department of Dermatology, Brigham and Women's Hospital and Harvard Medical School, 77 Avenue Louis Pasteur, HIM-660, Boston, MA 02115, USA
2 Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge, MA 02142, USA
3 Division of Gastroenterology and Nutrition, Children's Hospital and Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115, USA
4 Department of Immunology, University Medical Center Utrecht, Utrecht, The Netherlands

Correspondence to: M. Boes; E-mail: mboes{at}rics.bwh.harvard.edu


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Maturation of dendritic cells (DCs) regulates protein sorting in endosomal compartments to promote the surface expression of molecules involved in T cell activation. MHC Class II complexes are mobilized to the surface via intracellular effector molecules that remain largely unknown. We here show that protein kinase C (PKC) stimulates Class II antigen surface expression, using knock-in mice that express a Class II–green fluorescent protein fusion protein as a read out. Selective inhibition of PKC{delta} counteracts the ability of DCs to stimulate Class II MHC-restricted antigen-specific T cells. Activation of PKC does not affect antigen uptake, peptide loading and surface display of Class I MHC and transferrin receptor in DCs. We show that activation-induced Class II MHC surface expression is dependent on activation of PKC{delta} and conclude that this event is pivotal for optimal CD4 T cell activation.

Keywords: antigen presentation/processing, antigens/peptides/epitopes, dendritic cells, rodent


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
The initiation of a productive adaptive immune response requires the surface display of peptides bound to products of the MHC locus on antigen-presenting cells (APCs). Activation of naive T cells is accomplished most effectively when dendritic cells (DCs) display sufficient peptide–MHC complexes to T cells in the simultaneous presence of accessory molecules (1–3). Class II MHC molecules are expressed by professional APCs, in contrast to Class I MHC complexes which are more widely distributed (4). Class II MHC complexes acquire antigenic cargo for presentation to CD4 T cells by intersection of the endocytic and Class II biosynthetic pathways (5). The biosynthesis of Class II MHC and their mode of peptide acquisition are well established (6–9), while the mechanisms underlying the later stages of peptide–Class II trafficking are still poorly defined. Efficient display of peptide–Class II MHC complexes involves the proteolysis of both internalized protein antigen and Class II-associated invariant chain (Ii). During biosynthesis of Class II {alpha}–ß complexes, Ii associates with Class II dimers to achieve proper folding, occupying the peptide-binding cleft (10, 11). Recognition of targeting motifs in the Ii cytoplasmic domain guides trafficking of Ii–Class II MHC complexes from the endoplasmic reticulum via the trans-Golgi network to late endosomal antigen-processing compartments for binding of antigen-derived peptides (12). Peptide-loaded Class II MHC complexes can then be transported to the cell surface for inspection by appropriately restricted CD4 T cells.

DCs are highly effective at stimulating naive T cells in comparison to other APCs such as macrophages and B cells. Activation of DCs can lead them to undergo a process called maturation, for example when microbial products trigger surface receptors to pathogen-associated molecular patterns [i.e. Toll-like receptors (TLRs)] (13). During DC activation, endosomal sorting and trafficking of endosomal compartments are adjusted to favor peptide loading, as well as surface display of loaded peptide–Class II MHC complexes (14, 15). Other activation-mediated changes include down-regulation of endocytosis, up-regulation of chemokine receptors and induced formation of membrane extensions thought to optimize T cell activation (16). Cytoskeletal rearrangements, trafficking of membrane proteins and cell adhesion can all be regulated by members of the protein kinase C (PKC) family of serine–threonine protein kinases (17, 18). What are the mechanisms that mediate activation-induced surface display of Class II MHC complexes in DCs?

Members of the PKC family of serine–threonine kinases are activated when cells that express TLR4 are exposed to its ligand LPS (19, 20). We hypothesized that the activity of PKC family kinases is involved in up-regulating Class II MHC during DC activation. The observation that TLR4-mediated LPS signaling stimulates the transcription of IL-12 subunits p35 and p40 is in agreement with this notion (21, 22). Conversely, PKC activation is inhibited in macrophages derived from mice that carry a mutation in the TLR4 gene and that are therefore hyporesponsive to LPS (23). Endosomal trafficking of proteins can also be regulated by PKC activity, presumably by phosphorylation of serine–threonine residues near endosomal targeting motifs of the substrate (24–26). However, whether the activation-induced surface display of Class II MHC in DCs involves PKC activity has not been studied. We here describe the role of PKC activation in Class II antigen presentation by DCs, using bone marrow-derived DCs from Class II–green fluorescent protein (GFP) knock-in mice. We define PKC{delta} as the isoform responsible for stimulating Class II MHC plasma membrane expression.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Mice
Class II–GFP knock-in mice have been described (14, 27, 28). Class II–/– mice were obtained from Jackson Laboratories (29). All mice were backcrossed to C57Bl/6 for at least 10 generations. Mice were housed in a barrier facility and studies were performed according to the institutional guidelines for animal use and care.

DC culture and treatment
Bone marrow was extracted from the bone marrow cavity of murine femurs. The bone marrow was re-suspended until a single-cell suspension was generated. Cells were plated at 2.5 x 105 cells per well in 200 µl of DMEM/10% FCS/2 mM glutamine/200 U ml–1 penicillin/200 µg ml–1 streptomycin without phenol red and in the presence of 10 ng ml–1 granulocyte macrophage colony-stimulating factor (GM-CSF) (PeproTech, Rocky Hill, NJ, USA) and 1 ng ml–1 IL-4 (Roche Molecular Biochemicals, Somerville, NJ, USA). Bone marrow precursors were cultured for 4 days at 37°C in plastic dishes or cover slip dishes (Lab-Tek, to allow for live-cell confocal imaging) in a 5% CO2 and 95% air incubator. Inclusion of GM-CSF and IL-4 induces the selective outgrowth of CD11c+ Class II MHC+ DCs (14, 30, 31). The presence of elaborate endosomal compartments expressing large amounts of Class II–GFP was used as an additional DC marker. Where indicated, DCs at day 4 of culture were stimulated with 100 ng ml–1 LPS, 25 ng ml–1 phorbol 12-myristate 13-acetate (PMA) (Sigma–Aldrich), 500 ng ml–1 ionomycin (Sigma–Aldrich) or rottlerin (2 µM in 3-h experiments or 10 µM when analyzed after 18 h, Calbiochem). After culture at 37°C, DCs were harvested by vigorous re-suspension and transferred to 1.2 ml FACS tubes (USA Scientific) for staining for flow cytometry.

Flow cytometry
Harvested DCs were incubated 5 min on ice with Fc block 1:200 (BD Biosciences) in FACS buffer: PBS (GIBCO BRL) supplemented with 5% FCS. Immunostaining was performed for 15 min on ice with fluorophore-conjugated antibodies against CD11c, I-Ab, CD86, H2-Kb, CD4, CD69 and Vß5. All antibodies for flow cytometry were directly fluorophore-conjugated and were obtained from BD Biosciences. After immunostaining, the cells were pelleted by centrifugation (1200 x g, 5 min, 4°C) and washed three times with cold FACS buffer. FACS analysis was performed on a FACSCalibur flow cytometer (BD Biosciences).

Antigen endocytosis assay
DCs at day 4 of culture were treated with LPS, PMA and ionomycin or left untreated for 3 h at 37°C. Half of the DCs were then moved to 4°C and half the DCs were kept on 37°C, and FITC-conjugated ovalbumin (OVA) was supplemented for a further 30-min incubation (5 µg ml–1, Molecular Probes). After the 60 min of incubation at 4 or 37°C, DCs were washed and stained with anti-CD11c–APC antibody and immediately analyzed by flow cytometry as described above.

T cell activation assay
DCs at day 4 of culture were cultured in round-bottom 96-well plates in the presence of 0–40 µM OVA (Sigma) for 3 h in conjunction with various combinations of LPS, PMA and ionomycin (at the concentrations described above), and then washed three times with DC culture medium. In assays where rottlerin pre-treatment was done, DCs were pulsed with 4 µM OVA antigen O/N at day 3 of culture. At day 4, DCs were washed three times, rottlerin was added (10 µM) for 30 min and then LPS or PMA were added for 3 h. DCs were washed three times and OTII T cells (5 x 105) were added to the stimulated DC cultures for 4 or 18 h in normal media. At the end of the culture, the T cells were harvested and transferred to 1.2 ml FACS tubes on ice for antibody staining and flow cytometry analysis.

Pulse–chase analysis and immunoprecipitations
Pulse–chase experiments and immunoprecipitations were performed as described (32). Briefly, DCs were starved in cysteine/methionine-free media for 45 min and labeled for 45 min with 0.1 mCi ml–1 (35S) cysteine/methionine (PerkinElmer) in the presence of the indicated drug treatments. Cells were chased for the indicated times in complete DC media in the presence of drug treatments. Cells were lysed in NP-40 lysis buffer: 1% NP-40, 150 mM NaCl, 5 mM EDTA, 0.5 mM Tris, pH8.0 and complete protease inhibitor cocktail (Roche), pH 7.6. Lysates were pre-cleared with normal mouse serum together with Staph A. Class II MHC complexes were immunoprecipitated using an antibody to Class II molecules, N22 (33), and recovered with Staph A. After washing, the immunoprecipitates were split in half, re-suspended in sample buffer and incubated at room temperature, or boiled for 5 min. Samples were then analyzed by SDS–PAGE.

Reverse transcription–PCR
DCs were grown from mouse bone marrow as described above. Mouse cerebrum served as a control tissue in which most PKC isoforms are expressed (34). Cells were treated with DNAse to dissipate contaminating DNA. Template mRNA was extracted using an RNA extraction kit according to the manufacturer's recommendations (Ambion). The concentration of RNA was determined by densitometry by UV spectrofotometer (260 nm, Beckman). The amount of mRNA template used was 100 ng (cerebrum) or 250 ng (DCs). PKC primer sequences and annealing temperatures are described (34). Reverse transcription (RT)–PCR was done as recommended by the manufacturer (Stratagene). PCRs were allowed to proceed for 45 cycles (cerebrum) or 30 cycles (DCs).

Cell lysis and western blotting
Bone marrow-derived DCs (day 4 of culture) were re-suspended in cold lysis buffer: 50 mM Tris pH 7.5, 150 mM NaCl, 5 mM EDTA, 50 mM NaF, 1 mM Na3VO4 and complete protease inhibitor cocktail (Roche, 30 min, 4°C). Protein concentrations were normalized using the BCA Protein Assay Kit (Pierce) and were run on 4–20% SDS–PAGE gradient midigels (Bio-Rad). After transfer to polyvinylidene difluoride (PVDF) membrane (Bio-Rad), proteins were blocked for 1 h at 25°C with 5% milk block in Tris-buffered saline/0.1% Tween 20 (TBS-T) (Sigma). Membranes were blotted with mAbs specific to PKC isoforms {alpha}, ß, {delta} and {lambda} (BD Biosciences, 1:500) or with phospho-PKC{delta}/{theta} (Ser643/676) antibody (Cell Signaling Technology, Inc., 1:1000). After three washes with TBS-T (10 min each), membranes were incubated for 30 min with anti-mouse (for PKC{alpha}, ß, {delta} and {lambda} blots) or anti-rabbit (for anti-phospho PKC{delta}/{theta}) HRP-conjugated secondary antibodies (Jackson ImmunoResearch) (1:10 000 in 2% milk:TBS-T). Blots were developed with a chemiluminescent substrate (Bio-Rad) and x-ray film (Amersham Biosciences).

Immunofluorescence staining of mouse PKC{delta} in DCs
DCs were generated from bone marrow in cover slip bottom dishes (Lab-Tek II, Nalge Nunc) for 4 days. DCs were fixed using 4% PFA by adding an equal volume of PFA (8%, 37°C, 10 min) to cultured cells. DCs were permeabilized using 0.1% Triton-X in PBS (5 min, RT) and washed three times using PBS. Cells were then blocked using 10% BSA:PBS (1 h, RT) and stained 2 h using the anti-mouse PKC{delta} mAb (BD Biosciences, PKC Sampler Kit, 1:100 in 10% BSA:PBS) and developed using 1:400 secondary Alexa 568-conjugated goat anti-mouse antibody (Molecular Probes). Imaging was performed by spinning disc confocal microscopy at the microscopy center of the Department of Pathology, Harvard Medical School.

Statistical analyses
An unpaired two-tailed t-test was used for statistical analyses.

P-values below 0.05 are considered statistically significant and are indicated in the figure legends as *P < 0.05 or P < 0.01 where indicated and **P < 0.005.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
PKC stimulation induces surface display of Class II MHC in mouse DCs
To visualize whether activation of PKC is involved in the activation-induced surface display of Class II MHC complexes, we made use of bone marrow-derived DCs from Class II–GFP knock-in mice (14, 35, 36). Untreated, resting DCs were compared with DCs treated with PMA/ionomycin, a commonly used activator of conventional and novel forms of PKC. As a positive control for activation, we treated DCs from Class II–GFP mice with LPS (100 ng ml–1), in the presence or absence of PMA/ionomycin, and analyzed them by confocal microscopy (Fig. 1a). Untreated cells exhibit an immature phenotype, as characterized by the presence of the majority of Class II MHC in endosomal compartments (Fig. 1a). After 3 h of LPS treatment, surface display of Class II MHC is seen (Fig. 1a, top panels) (37, 38). However, combined treatment of LPS/PMA/ionomycin shows rather different kinetics of activation. Robust surface display of Class II MHC can be visualized already after 1 h of treatment, further enhanced after a 3 h of treatment (Fig. 1a, bottom panels). This rapid activation is also evident by acquisition of a mature morphological phenotype of the DCs (39, 40).


Figure 1
View larger version (26K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1. PKC agonists rapidly induce Class II MHC surface display and significant changes in DC morphology. (a) Bone marrow-derived DCs (day 4 of culture in the presence of GM-CSF and IL-4) were cultured in cover slip dishes and analyzed by confocal microscopy after 0, 1 and 3 h of treatment with LPS alone (top panels) or LPS, PMA and ionomycin (bottom panels). (b) Increased surface display of Class II MHC upon treatment with PMA. Bone marrow-derived DCs (day 4 of culture) were cultured for 3 h in the presence of LPS, ionomycin and PMA or left untreated. Treatment with PMA alone stimulates both Class II surface display and acquisition of mature DC morphology. Images are representative of three independent experiments.

 
Conventional PKC isoforms ({alpha}, ßI, ßII and {gamma}) require both diacyl glycerol (DAG) (or phorbol ester) and a rise in cytosolic calcium ions (Ca2+) for their activation, while the novel PKC isoforms {delta}, {epsilon}, {tau} and {eta} are DAG dependent but Ca2+ independent (41–44). Atypical PKC isoforms {zeta} and {lambda} appear regulated independently of DAG and Ca2+ fluxes (42). To investigate whether a conventional or novel PKC isoform is involved in stimulating the translocation of Class II MHC complexes to the plasma membrane, we treated DCs with PMA or ionomycin separately or with LPS as control. During the last 5 min of culture, Alexa 594-conjugated cholera toxin B subunit was added to stain ganglioside (GM1)-containing lipid domains (45) and so facilitate the visualization of Class II MHC expression at the cell surface. Untreated DCs have low levels of Class II MHC complexes at the cell surface and an immature morphology (Fig. 1b). After 3 h of stimulation, LPS- or ionomycin-treated DCs generally express modest surface levels of Class II MHC. PMA treatment induces surface display of Class II MHC, as visualized by co-localization of Class II MHC (GFP, green) with the GM1-containing lipid domains in the plasma membrane (Alexa 594, red) (yellow color, Fig. 1b). Since PMA treatment in the absence of ionomycin induces Class II MHC surface display, we conclude that activation of novel PKC isoforms induces surface display of Class II MHC in mouse DCs.

PKC{delta} is expressed in mouse DCs
Most cells express multiple PKC isoforms and therefore attribution of a specific function to a specific isoform is not straight forward (46). To determine which PKC isoforms are expressed in mouse bone marrow-derived DCs, we extracted mRNA from DCs and from mouse cerebrum as a control. RT–PCR was performed using primer sequences (34) for individual PKC isoforms. In mouse cerebrum, many PKC isoforms are expressed (34): we detected transcripts of PKC{alpha}, ß, {delta}, {epsilon}, {theta}, {zeta}, {lambda} and {gamma} (Fig. 2a, top, left). Using mRNA from DCs as template, we detected transcripts of PKC{alpha}, ß, {delta} and {lambda} (Fig. 2a, top, right; asterisks indicate the presence of PKC expression), but not {gamma}, {epsilon}, {theta} and {zeta}. PKC{alpha} and ß are conventional isoforms and can thus be excluded as candidates responsible for a PMA-induced process (34, 41). PKC{lambda} is an atypical PKC isoform, which is not stimulated by PMA treatment either (42). As a novel isoform, which can be activated by PMA treatment alone (42), PKC{delta} must therefore be responsible for the observed rapid Class II MHC surface display.


Figure 2
Figure 2
View larger version (50K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2. PKC{delta} is expressed in mouse bone marrow-derived DCs and is activated upon PMA stimulation. (a) DCs (day 4 of culture in the presence of GM-CSF and IL-4) were purified using CD11c-based MACS purification (Miltenyi). RT–PCR analysis of lysates from mouse cerebrum (left) and purified mouse bone marrow DCs (right). Asterisks indicate the lanes where an RT–PCR product was seen (PKC{alpha}, ß, {delta} and {lambda}). Experiment is representative of four experiments with similar outcome. DC lysates were also analyzed by western blot, after SDS–PAGE and protein transfer to PVDF membrane, using mAbs to PKC{alpha}, ß, {delta} and {lambda}. Only PKC{delta} was detected, as indicated by an asterisk. (b) DCs were grown in glass cover slip dishes, fixed using 4% PFA, permeabilized and stained using unconjugated antibodies against PKC isoforms PKC{alpha}, PKCß, PKC{delta} and PKC{lambda}, followed by Alexa 568-conjugated secondary antibody. Day 4 Class II–GFP-positive DCs as well as Class II–GFP-negative bone marrow cells appear to express PKC{delta} only, and not the three other PKC isoforms analyzed. (c) DCs were stimulated with PMA for 0, 1, 5, 30 and 180 min and were lysed. lysates were run on SDS–PAGE and blotted to PVDF membrane, and were blotted using phospho-PKC{delta}/{theta} (ser643/676) antibody. Activation-induced phospho-PKC{delta} protein was detected most prominently at 30 min after PMA treatment. PKC{theta} is not detected in mouse DCs. Experiment shown is representative of three independent experiments. (d) DCs were grown in cover slip dishes, treated with LPS, ionomycin and PMA or left untreated (3 h, 37°C), and then fixed, permeabilized and stained using PKC{delta} antibody followed by Alexa 568-conjugated secondary antibody. DCs were analyzed by confocal microscopy (x100 objective, Z-stack analysis, 1 µm step size). Experiment shown is representative of four independent experiments.

 
To determine at the protein level whether PKC{alpha}, ß, {delta} or {lambda} are expressed in mouse DCs, we performed western blot analysis using antibodies specific to these four PKC isotypes. Whereas PKC{delta} was readily observed, PKC{alpha}, ß and {lambda} were not detected by western blot (Fig. 2a, bottom). To confirm the western blot data, immunofluorescence stainings for PKC{alpha}, ß, {delta} and {lambda} were performed on fixed and permeabilized DCs. Secondary Alexa 568-conjugated antibody was used for visualization of the PKC isoforms (Fig. 2b). Also using immunofluorescence microscopy, only the PKC{delta} isoform was detected. Taken together, only PKC{delta} protein is prevalent in mouse DCs.

Acquisition of PKC{delta} biological activity upon phorbol ester treatment
To establish whether PKC{delta} is activated upon phorbol ester treatment, we performed western blot and immunofluorescence microscopy studies. The enzymatic activity and biological function of PKC{delta} is controlled by autophosphorylation of serine 643 of PKC{delta} (30), which can be measured by western blot. DCs were treated with PMA for the indicated time points and were lysed. Lysates were run on SDS–PAGE and transferred to PVDF for blotting with an antibody specific to phospho-PKC{delta}/{theta} (serine 643/676). This antibody detects endogenous levels of PKC{delta} only when phosphorylated at serine 643 (and PKC{theta} only when phosphorylated at serine 676). Already at time point zero, at which time PMA was added, some PKC{delta} was phosphorylated. The level of phosphorylated PKC{delta} increased over time after PMA treatment, and reached an optimum at 30 min. Antibody reactivity to Glyceraldehyde 3-phosphate Dehydrogenase was used as a loading control, unaffected by PMA treatment (Fig. 2c). We next visualized the intracellular localization of PKC{delta} in maturing DCs, confocal microscopy was performed on Class II–GFP DCs (green) stained with antibody to PKC{delta} (red) (Fig. 2d). DCs were left untreated or cultured with LPS, PMA and ionomycin for 3 h, and then fixed in 4% PFA and stained with anti-PKC{delta} mAb (BD Biosciences). Untreated DCs showed a predominantly round or elongated morphology with most Class II MHC in endosomes and PKC{delta} in the cytosol. DCs treated with LPS or ionomycin exhibited a predominantly elongated morphology, with a modest surface level of Class II MHC and cytosolic localization of PKC{delta}. In contrast, PMA-treated DCs displayed a mature phenotype, with dendritic extensions and increased surface levels of Class II MHC. Moreover, PMA treatment induced the transfer of PKC{delta} to the plasma membrane and nuclear envelope in DCs (Fig. 2d). Our results show that PMA treatment induces also the membrane translocation of PKC{delta}, indicative for acquiring its biological function.

PKC stimulation of DCs induces increased surface levels of I-Ab, but not of CD86, H2-Kb or transferrin receptor
PKC{delta} activation induces surface-directed trafficking of Class II MHC and of itself. To study the specificity of this event, we treated DCs with LPS, PMA or ionomycin for 3 h and measured the surface display of Class II MHC (I-Ab), CD86, Class I MHC (H2-Kb) and transferrin receptor (TfR) by flow cytometry (Fig. 3). Changes in surface display of I-Ab, CD86, Kb or TfR after treatment with LPS, PMA or ionomycin are shown as a percentage change to untreated DCs. Class II MHC surface expression was significantly induced by LPS, PMA or ionomycin (40–50% increase compared with untreated DCs, P < 0.005, Fig. 3), while confocal imaging had indicated that PMA treatment (but not LPS or ionomycin) induced the most dramatic Class II MHC surface display associated with cytoskeletal rearrangements (Fig. 1b). PMA or ionomycin were unable to induce surface expression of CD86, while LPS treatment did induce significant CD86 up-regulation, as expected (47, 48). Class I MHC was not induced by 3-h stimulation with LPS, PMA or ionomycin, and TfR surface expression was unaffected by LPS or ionomycin treatment. We consistently found that PMA treatment induced significant internalization of TfR, possibly reflecting the internalization of TfR into recycling endosomes (49, 50). Taken together, our data suggest a specific role for PKC{delta} activation in stimulating the surface display of Class II MHC as opposed to Class I MHC or CD86 trafficking.


Figure 3
View larger version (20K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3. PKC activation stimulates surface display of Class II MHC, but not of Class I MHC, CD86 or transferrin receptor. Bone marrow-derived DCs were gated based on high expression of Class II–GFP. DCs were analyzed by flow cytometry analysis of surface display of (a) Class II MHC (I-Ab), (b) CD86, (c) Class I MHC (H2-Kb) and (d) transferrin receptor (TfR). Surface expression of H2-Kb, CD86 and TfR are not affected by treatment with PKC agonists, suggesting that LPS and PMA selectively stimulate a surface-directed endosomal pathway, rather than generalized vesicle exocytosis. Values are mean ± SE of at least six experiments (*P < 0.05; **P < 0.005).

 
PKC stimulation of DCs increases Class II antigen presentation to antigen-specific T cells
Is PKC{delta} involved in enhancing Class II MHC antigen presentation to antigen-specific T cells under conditions of limited antigen delivery? DCs were treated with 0, 4 and 40 µM OVA for 3 h, in the presence of LPS, PMA and/or ionomycin. DCs were then washed three times prior to addition of OVA-specific naive T cells from transgenic mice that express an OVA-specific I-Ab-restricted TCR (OTII). After 4 and 18 h of co-culture of T cells with DCs, T cells were analyzed for their activation status by flow cytometry (CD4–peridinin chlorophyl protein, (PerCP). Vß5–PE and CD69–FITC, Fig. 4). In the absence of OVA, background levels of T cell activation were seen (except when LPS, PMA and ionomycin were all present, we have not investigated this further). DCs treated for 4 h with 4 µM OVA alone induced activation of 3% of OTII T cells, which increased to 50% when DCs were treated in addition with phorbol ester (Student's t-test, P < 0.005 comparing PMA-treated with untreated sample). Comparable data were obtained when DCs were treated with a higher concentration of OVA (40 µM) or when T cell activation was allowed to continue for 18 h instead of 4 h (Fig. 4). Treatment with 40 µM resulted in full activation of most T cells by 18 h. Taken together, we conclude that PKC stimulation enhances the ability of DCs to stimulate Class II MHC-restricted T cells under conditions of limited antigen availability.


Figure 4
View larger version (17K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4. PKC activation potentiates antigen-specific T cell activation by antigen-laden DCs. (a) Wild-type DCs were treated for 4 h with 4 µM OVA alone or in combination with LPS, PMA or ionomycin, and then washed three times and co-cultured for 4 h with naive OTII T lymphocytes. Activation of OTII T cells was determined by measuring CD69 up-regulation by flow cytometry. (b) As in Fig. 5(a), DCs from wild-type mice were treated with 40 µM OVA alone, or in conjunction with LPS, PMA, ionomycin or combinations thereof. DCs were then washed three times and co-cultured for 4 h with naive OTII T lymphocytes. Activation of OTII T cells was determined by flow cytometry by analysis of up-regulation of the early activation marker CD69. Significant differences between the mean values of T cell activation by LPS-, iono- and PMA-treated DCs are indicated by asterisks (*P < 0.05; **P < 0.005).

 
Antigen uptake in DCs is independent of PKC activity
Kinases of the PKC family play an important regulatory role in actin reorganization in many cell types, and actin polymerization is involved in the early phases of phagocytosis (51, 52). We therefore investigated whether PKC{delta} is involved in antigen presentation through mediating antigen uptake. Wild-type DCs were treated with LPS, PMA or ionomycin for 3 h at 37°C (or at 4°C as control), and fluorescein-conjugated OVA was added for one additional hour of culture at 37°C (or at 4°C as control). Uptake was determined by flow cytometry using a marker for DCs (CD11c–APC) and measuring OVA–FITC fluorescence within DCs (Fig. 5). Ionomycin treatment stimulated the uptake of OVA, whereas LPS pre-treatment significantly decreased internalization of OVA by DCs, consistent with induction of TLR-induced DC maturation (53). No significant difference was found in uptake of OVA by 3-h pre-treatment with PMA. When OVA–FITC was added simultaneously with LPS, PMA or ionomycin for 3 h, no difference was seen either (data not shown). We conclude that PKC{delta} does not promote Class II antigen presentation by stimulating antigen uptake by DCs.


Figure 5
View larger version (42K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5. PKC activation does not stimulate antigen uptake by DCs. Wild-type DCs were incubated with LPS, PMA and ionomycin for 3 h, and then FITC-conjugated OVA (5 µg ml–1) was added for 1 h of uptake at 4 or 37°C. Background level of FITC–OVA in CD11c+ cells at 4°C was below 5% for all samples. FITC fluorescence was plotted against the DC marker CD11c, showing the percentage of CD11c-positive cells that endocytosed FITC–OVA. A representative experiment is shown out of a total of five independent experiments, each performed in triplicate.

 
Comparable level of peptide loading onto Class II MHC molecules
To determine whether PKC stimulates the rate of intracellular Class II MHC transport and peptide acquisition, we performed pulse–chase analysis and examined the SDS resistance of Class II MHC molecules as a measure of peptide loading. DCs were metabolically labeled with 35S-cysteine/methionine for 45 min and chased for 0, 1, 2, 3 and 6 h. Drug treatments were included during the pulse and chase. After the indicated chase points, cell lysates were prepared and correctly folded {alpha}ß–peptide complexes were immunoprecipitated using a conformation-specific anti-Class II antibody, N22. Immunoprecipitates were resolved by 12.5% SDS–PAGE (Fig. 6). Immediately after pulse labeling, stable peptide–Class II dimers were not yet formed in either of the DC samples. At the chase time points analyzed, regardless of drug treatment, peptide-loaded stable Class II dimers were detected at 1 h and were most prominent at 3 and 6 h. Regardless of the presence of PMA, ionomycin or LPS, at 6 h of chase, full-length Ii was no longer immunoprecipitated, indicating that all labeled N22-immunoprecipiated Class II MHC had acquired peptide. We conclude that PKC{delta} does not affect Class II biosynthesis and peptide acquisition, as assessed by this method.


Figure 6
View larger version (69K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6. PKC activation does not stimulate the formation of peptide–Class II MHC complexes in DCs. Wild-type DCs were pulsed with 35S-cysteine/methionine for 45 min and chased for 0, 1, 2, 3 and 6 h in the presence of PMA, ionomycin or LPS. Class II MHC molecules were immunoprecipitated using conformation-specific antibody, N22, incubated for 5 min at room temperature or boiled and separated on SDS–PAGE. {alpha}ßIi: Ii-loaded Class II; {alpha}ßp: stable Class II dimers of {alpha}ß–peptide; {alpha}: alpha chain of Class II; Ii: full-length Ii and ß: beta chain of Class I MHC. A representative experiment of two is shown.

 
Treatment with rottlerin reduces surface display of Class II MHC
If activation of PKC{delta} is indeed involved in activation-induced Class II surface display in DCs, conversely, an inhibitor of PKC{delta}, rottlerin, should inhibit expression of Class II MHC at the cell surface (54, 55). When used in high concentrations (20 µM), rottlerin can inhibit other protein kinases beside PKC{delta} (56). We therefore used rottlerin only in short-term treatments and at concentrations of 2–5 µM. DCs were pre-treated with rottlerin (2 µM, 30 min, 37°C) after which LPS or PMA was added or cells were left untreated for 3 h of culture. DCs were then stained on ice using antibodies to CD11c (–APC) and I-Ab (–PE) and analyzed by flow cytometry (Fig. 7a). Treatment with rottlerin resulted in a significant decrease in Class II surface display (decrease of 20%, P < 0.01 comparing rottlerin-treated with untreated DCs). DCs cultured in the presence of PMA showed an increase in Class II surface display that was inhibited by pre-treatment with rottlerin (P < 0.01, comparing PMA treatment with rottlerin–PMA treatment). Three hours of LPS treatment induced only a modest increase in Class II MHC, and rottlerin-mediated inhibition of LPS-dependent Class II up-regulation was not observed (Fig. 7a). This set of experiments differentiates between a LPS-dependent pathway and a PKC-mediated pathway of Class II surface export.


Figure 7
View larger version (13K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 7. Selective inhibition of PKC{delta} counteracts surface display of Class II MHC in DCs and antigen-specific CD4 T cell activation. (a) DCs were pre-treated with rottlerin or left untreated, followed by LPS or PMA treatment for 3 h. Surface display of Class II MHC was measured by flow cytometry using PE-conjugated antibody to I-Ab. Rottlerin pre-treatment counteracts PMA-induced surface display of Class II MHC (*P < 0.01). Experiment shown is representative of four independent experiments. (b) DCs from wild-type and MHC Class II–/– mice were cultured in the presence of OVA O/N. DCs were washed twice, pre-treated with rottlerin or left untreated for 30 min, and then LPS or PMA was added where indicated (3 h, 37°C). DCs were washed three times and OTII T cells were added (18 h, 37°C). The effect of rottlerin, LPS and PMA on T cell activation was assayed by flow cytometry measuring up-regulation of CD25–APC on OTII T cells, gated on CD4–PE–Cy7 and Vß5–PE). Class II–/– DCs do not stimulate T cell activation under any of the conditions. Experiment shown is representative of three experiments that were performed.

 
Treatment with rottlerin counteracts T cell activation by DCs
We next investigated the consequences of inhibition of PKC{delta} in functional antigen presentation assays. DCs were pulsed O/N with 4 µM OVA, washed and then pre-treated with rottlerin (5 µM, 30 min, 37°C) after which LPS or PMA was added for 3 h. DCs were washed and naive OTII T cells were added for 18 h of co-culture in the absence of stimuli or inhibitors (Fig. 7b). T cell activation was measured by up-regulation of CD25 (and CD69, data not shown), by flow cytometry. DCs from Class II–/– mice (29) were included in this assay as controls: OVA-treated Class II–/– DCs induced activation in <4% of OTII T cells, thus excluding the possibility that T cells are stimulated by carry over of PMA.

OVA-loaded wild-type DCs induced T cell activation in 38% of OTII T cells, either when DCs were left untreated or when LPS was included (Fig. 7b). In this experimental setup, the known stimulatory ability of LPS in DC function was not evident. Treatment of OVA-laden DCs with rottlerin (3.5 h, 37°C) resulted in a decrease in T cell activation to ~8% of T cells (Fig. 7b). Three-hour PMA treatment of OVA-loaded DCs resulted in activation of 90% of T cells, which was decreased to 20% by 30-min pre-treatment with rottlerin. Comparable data were obtained for CD69 up-regulation (data not shown). Taken together, these experiments confirm a stimulatory role for PKC{delta} in Class II MHC-mediated T cell activation.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Bone marrow-derived DCs that express fusion proteins of Class II and GFP are now commonly used to study intracellular trafficking of peptide–Class II complexes (14, 15, 35, 36, 57). We here show that PKC{delta} is pivotal in surface-directed transport of peptide–Class II MHC to the cell surface. Stimulation of PKC{delta} promotes the surface expression of Class II MHC on the cell surface and Class II-mediated T cell activation. Conversely, inhibition of PKC{delta} counteracts the ability of DCs to activate naive Class II-restricted T lymphocytes.

A role for PKC in Class II MHC-mediated antigen presentation in mouse DCs had not been studied. Different PKC isoforms are involved in exocytosis and endosomal trafficking, as was demonstrated in various other cell types. In CTL, PMA/ionomycin treatment triggers the exocytosis of cytolytic granules (58, 59). Specifically, microtubule-organizing center polarization toward the CTL–target cell interphase was suggested to require PKC phosphorylation of microtubule-associated proteins or of kinesin or dynamin motors (59). Where Class II MHC antigen presentation is concerned, the role of PKC family kinases was investigated earlier in B cell tumor lines. Ligation of the BCR induces a signal transduction cascade via the Ig{alpha} and Igß co-receptors, and results in a cascade of protein tyrosine kinase activation and in the production of second messengers such as IP3 and DAG (60). In B cells, protein tyrosine kinase pathways and DAG-induced PKC activation converge at the level of the mitogen-activated protein kinase pathway, leading to gene transcription of genes involved in B cell activation (61, 62). PKC activation by PMA treatment of B lymphoma cells delayed the degradation of Class II-associated Ii and resulted in accumulation of Ii intermediates, equivalent to that seen in leupeptin-treated cells (63). Consequently, PMA treatment impaired the formation of SDS-stable dimers and peptide/Class II presentation in mouse B cells for some T cell epitopes. Thus, PKC activation through PMA treatment reduces peptide/Class II presentation in mouse B cells (63). Our study in mouse DCs now shows that in DCs, PKC activation supports Class II presentation by stimulating Class II MHC surface display rather than inhibiting peptide–Class II dimer formation as seen in mouse B cells. In human B cell lines, PKC directly phosphorylates the Class II-associated Ii, which enhanced the kinetics of Ii degradation and Class II presentation (64). Since Ii is not phosphorylated in murine APCs (63) as it is in humans (64), little effect of PKC activity is expected on Class II biosynthesis and peptide loading in mouse DCs. Our results (Fig. 6) are in line with this conclusion, as confirmed by the demonstration of unchanged kinetics of the formation of SDS-stable dimers.

From studies in multiple cell types it was deduced that the different PKC isoforms are not functionally redundant but mediate discrete biological effects (42, 65). It is therefore important to demonstrate a role for a particular PKC isoform in mediating a specific cell biological process. PKC family members are involved in DC lineage commitment from human hematopoietic progenitor cells or monocytes (66, 67). These differentiation processes require the presence of Ca2+ and DAG and are mediated conventional PKC family members PKC{alpha} and PKCß (68–70). In DCs, Class II MHC-mediated antigen presentation was strongly enhanced by phorbol ester treatment alone, which suggested the involvement of a novel rather than conventional PKC isoform. Mouse DCs only express one novel PKC isoform, PKC{delta}. To directly show that PKC{delta} is involved in antigen presentation via Class II MHC molecules, we initially considered experiments involving retrovirus-mediated RNAi knockdown of PKC{delta} in primary bone marrow-derived DCs, but this method proved problematic for technical reasons. We refrained from using PKC{delta} knock-out mice (71) as PKC isoforms are involved in monocyte or DC development (70, 72, 73). Instead, we made use of rottlerin, a selective inhibitor to PKC{delta}, which we administered to wild-type end-differentiated DCs. Pre-treatment with rottlerin followed by LPS or PMA stimulation strongly counteracted the ability of DCs to activate naive T cells. PKC{delta} activity is thus a possible point of interference for MHC Class II-dependent antigen presentation.

Previous studies have addressed a role for PKC{delta} in DC biology. Apoptosis of mature human DCs as mediated by ligation of Class II MHC is regulated by activation of PKC{delta} (74). The same laboratory showed that DC maturation induced the recruitment of PKC{delta} to Class II MHC-positive lipid microdomains in the plasma membrane (75). Culture of human and murine DCs in the presence of bryostatin-1, an anti-neoplastic agent, with calcium ionophore induces DC maturation (76). Bryostatin or PMA/calcium ionophore induced DC-mediated proliferation of allogenic T cells and antigen-specific T cells. Moreover, bryostatin-stimulated T cell activation by DCs was strongly inhibited by pre-incubation with the pan-PKC inhibitor bisindolylmaleimide I or with the PKC{delta}-selective inhibitor rottlerin (76). Thus, this study already suggested that PKC{delta} activation under some circumstances stimulate Class II MHC-mediated T cell activation, a mechanism that we investigated here in further detail.

Microbe-derived signals are potent adjuvants that stimulate antigen presentation by DCs (77). LPS can do so via several mechanisms. LPS treatment transiently enhances antigen uptake (78), following which endocytosis is down-regulated (53, 79). LPS treatment stimulates the degradation of endocytosed antigen (80) and regulates endosomal sorting with the outcome that Class II MHC complexes relocate from internal vesicular membranes of multivesicular bodies to the H2-DM-containing delimiting membranes of Class II-positive endosomal compartments (37). The formation of tubular endosomes for surface-directed trafficking of peptide–Class II complexes is stimulated by the presence of LPS as well (15, 36, 81). Our data indicate that PKC{delta}-mediated stimulation of Class II antigen presentation is neither mediated by increased endocytosis nor by peptide loading. Others had shown that PKC activation in smooth muscle cells and neuronal growth cones can induce an outgrowth of microtubules from the perinuclear region into the cytoplasm, suggested to be involved in redistribution of vesicular trafficking along microtubules (82, 83). The trafficking of Class II MHC complexes from endosomal structures to the cell surface is also based on microtubule-dependent mechanisms (84, 85). Our findings that phorbol ester treatment induces robust Class II surface display and the appearance of dendritic extensions suggest that PKC{delta} activity stimulates Class II-mediated antigen presentation via induced surface-directed microtubule-based trafficking of Class II MHC complexes. In conclusion, activation of PKC{delta} potentiates DC function, by stimulating the display of peptide–Class II complexes at the DC plasma membrane.


   Acknowledgements
 
We thank the members of the Boes Laboratory for helpful discussions and critical reading of the manuscript. Funding source: Grant support is acknowledged from the Boehringer Ingelheim Fonds (F.C.M.S.), the National Science Foundation (to A.M.P.), the Harvard Skin Disease Research Center (M.B.) and the Netherlands organization for scientific research (NWO) (M.B.).


   Abbreviations
 
APC, antigen-presenting cell
Ca2+, calcium ion
DAG, diacyl glycerol
DC, dendritic cell
GFP, green fluorescent protein
GM1, ganglioside M1
GM-CSF, granulocyte macrophage colony-stimulating factor
Ii, invariant chain
OVA, ovalbumin
PKC, protein kinase C
PMA, phorbol 12-myristate 13-acetate
PVDF, polyvinylidene difluoride
RT, reverse transcription
TBS, Tris-buffered saline
TfR, transferrin receptor
TLR, Toll-like receptor
TBS-T, Tris-buffered saline Tween 20

   Notes
 
Transmitting editor: C. Terhorst

Received 6 November 2006, accepted 5 March 2007.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Vogt AB, Spindeldreher S, Kropshofer H. Clustering of MHC-peptide complexes prior to their engagement in the immunological synapse: lipid raft and tetraspan microdomains. Immunol. Rev. (2002) 189:136.[CrossRef][Web of Science][Medline]
  2. Krogsgaard M, Huppa JB, Purbhoo MA, Davis MM. Linking molecular and cellular events in T-cell activation and synapse formation. Semin. Immunol. (2003) 15:307.[CrossRef][Web of Science][Medline]
  3. Boes M, Ploegh HL. Translating cell biology in vitro to immunity in vivo. Nature (2004) 430:264.[CrossRef][Medline]
  4. Tortorella D, Gewurz BE, Furman MH, Schust DJ, Ploegh HL. Viral subversion of the immune system. Annu. Rev. Immunol. (2000) 18:861.[CrossRef][Web of Science][Medline]
  5. Peters PJ, Neefjes JJ, Oorschot V, Ploegh HL, Geuze HJ. Segregation of MHC class II molecules from MHC class I molecules in the Golgi complex for transport to lysosomal compartments. Nature (1991) 349:669.[CrossRef][Medline]
  6. Cresswell P. Assembly, transport, and function of MHC class II molecules. Annu. Rev. Immunol. (1994) 12:259.[CrossRef][Web of Science][Medline]
  7. Germain RN. MHC-dependent antigen processing and peptide presentation: providing ligands for T lymphocyte activation. Cell (1994) 76:287.[CrossRef][Web of Science][Medline]
  8. Pierre P, Mellman I. Developmental regulation of invariant chain proteolysis controls MHC class II trafficking in mouse dendritic cells. Cell (1998) 93:1135.[CrossRef][Web of Science][Medline]
  9. Bryant P, Ploegh H. Class II MHC peptide loading by the professionals. Curr. Opin. Immunol. (2004) 16:96.[CrossRef][Web of Science][Medline]
  10. Bijlmakers MJ, Benaroch P, Ploegh HL. Mapping functional regions in the lumenal domain of the class II-associated invariant chain. J. Exp. Med. (1994) 180:623.[Abstract/Free Full Text]
  11. Ghosh P, Amaya M, Mellins E, Wiley DC. The structure of an intermediate in class II MHC maturation: CLIP bound to HLA-DR3. Nature (1995) 378:457.[CrossRef][Medline]
  12. Bakke O, Dobberstein B. MHC class II-associated invariant chain contains a sorting signal for endosomal compartments. Cell (1990) 63:707.[CrossRef][Web of Science][Medline]
  13. Akira S, Takeda K, Kaisho T. Toll-like receptors: critical proteins linking innate and acquired immunity. Nat. Immunol. (2001) 2:675.[CrossRef][Web of Science][Medline]
  14. Boes M, Cerny J, Massol R, et al. T-cell engagement of dendritic cells rapidly rearranges MHC class II transport. Nature (2002) 418:983.[CrossRef][Medline]
  15. Chow A, Toomre D, Garrett W, Mellman I. Dendritic cell maturation triggers retrograde MHC class II transport from lysosomes to the plasma membrane. Nature (2002) 418:988.[CrossRef][Medline]
  16. Banchereau J, Briere F, Caux C, et al. Immunobiology of dendritic cells. Annu. Rev. Immunol. (2000) 18:767.[CrossRef][Web of Science][Medline]
  17. Keenan C, Kelleher D. Protein kinase C and the cytoskeleton. Cell. Signal. (1998) 10:225.[CrossRef][Web of Science][Medline]
  18. Newton AC. Regulation of protein kinase C. Curr. Opin. Cell Biol. (1997) 9:161.[CrossRef][Web of Science][Medline]
  19. Hu J, Jacinto R, McCall C, Li L. Regulation of IL-1 receptor-associated kinases by lipopolysaccharide. J. Immunol. (2002) 168:3910.[Abstract/Free Full Text]
  20. Noubir S, Hmama Z, Reiner NE. Dual receptors and distinct pathways mediate interleukin-1 receptor-associated kinase degradation in response to lipopolysaccharide. Involvement of CD14/TLR4, CR3, and phosphatidylinositol 3-kinase. J. Biol. Chem. (2004) 279:25189.[Abstract/Free Full Text]
  21. Kadowaki N, Ho S, Antonenko S, et al. Subsets of human dendritic cell precursors express different toll-like receptors and respond to different microbial antigens. J. Exp. Med. (2001) 194:863.[Abstract/Free Full Text]
  22. Weinmann AS, Mitchell DM, Sanjabi S, et al. Nucleosome remodeling at the IL-12 p40 promoter is a TLR-dependent, Rel-independent event. Nat. Immunol. (2001) 2:51.[CrossRef][Web of Science][Medline]
  23. Shinji H, Akagawa KS, Yoshida T. LPS induces selective translocation of protein kinase C-beta in LPS-responsive mouse macrophages, but not in LPS-nonresponsive mouse macrophages. J. Immunol. (1994) 153:5760.[Abstract]
  24. Dietrich J, Hou X, Wegener AM, Geisler C. CD3 gamma contains a phosphoserine-dependent di-leucine motif involved in down-regulation of the T cell receptor. EMBO J. (1994) 13:2156.[Web of Science][Medline]
  25. Pitcher C, Honing S, Fingerhut A, Bowers K, Marsh M. Cluster of differentiation antigen 4 (CD4) endocytosis and adaptor complex binding require activation of the CD4 endocytosis signal by serine phosphorylation. Mol. Biol. Cell (1999) 10:677.[Abstract/Free Full Text]
  26. Guinamard R, Signoret N, Ishiai M, Marsh M, Kurosaki T, Ravetch JV. B cell antigen receptor engagement inhibits stromal cell-derived factor (SDF)-1alpha chemotaxis and promotes protein kinase C (PKC)-induced internalization of CXCR4. J. Exp. Med. (1999) 189:1461.[Abstract/Free Full Text]
  27. Bikoff EK, Huang LY, Episkopou V, van Meerwijk J, Germain RN, Robertson EJ. Defective major histocompatibility complex class II assembly, transport, peptide acquisition, and CD4+ T cell selection in mice lacking invariant chain expression. J. Exp. Med. (1993) 177:1699.[Abstract/Free Full Text]
  28. Shi GP, Villadangos JA, Dranoff G, et al. Cathepsin S required for normal MHC class II peptide loading and germinal center development. Immunity (1999) 10:197.[CrossRef][Web of Science][Medline]
  29. Madsen L, Labrecque N, Engberg J, et al. Mice lacking all conventional MHC class II genes. Proc. Natl Acad. Sci. USA (1999) 96:10338.[Abstract/Free Full Text]
  30. Li W, Zhang J, Bottaro DP, Pierce JH. Identification of serine 643 of protein kinase C-delta as an important autophosphorylation site for its enzymatic activity. J. Biol. Chem. (1997) 272:24550.[Abstract/Free Full Text]
  31. Inaba K, Inaba M, Romani N, et al. Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. J. Exp. Med. (1992) 176:1693.[Abstract/Free Full Text]
  32. Villadangos JA, Riese RJ, Peters C, Chapman HA, Ploegh HL. Degradation of mouse invariant chain: roles of cathepsins S and D and the influence of major histocompatibility complex polymorphism. J. Exp. Med. (1997) 186:549.[Abstract/Free Full Text]
  33. Metlay JP, Witmer-Pack MD, Agger R, Crowley MT, Lawless D, Steinman RM. The distinct leukocyte integrins of mouse spleen dendritic cells as identified with new hamster monoclonal antibodies. J. Exp. Med. (1990) 171:1753.[Abstract/Free Full Text]
  34. Gangeswaran R, Jones KT. Unique protein kinase C profile in mouse oocytes: lack of calcium-dependent conventional isoforms suggested by rtPCR and Western blotting. FEBS Lett. (1997) 412:309.[CrossRef][Web of Science][Medline]
  35. Bertho N, Cerny J, Kim YM, Fiebiger E, Ploegh H, Boes M. Requirements for T cell-polarized tubulation of class II+ compartments in dendritic cells. J. Immunol. (2003) 171:5689.[Abstract/Free Full Text]
  36. Boes M, Bertho N, Cerny J, Op Den Brouw M, Kirchhausen T, Ploegh H. T cells induce extended class II MHC compartments in dendritic cells in a Toll-like receptor-dependent manner. J. Immunol. (2003) 171:4081.[Abstract/Free Full Text]
  37. Kleijmeer M, Ramm G, Schuurhuis D, et al. Reorganization of multivesicular bodies regulates MHC class II antigen presentation by dendritic cells. J. Cell Biol. (2001) 155:53.[Abstract/Free Full Text]
  38. Wilson NS, El-Sukkari D, Belz GT, et al. Most lymphoid organ dendritic cell types are phenotypically and functionally immature. Blood (2003) 102:2187.[Abstract/Free Full Text]
  39. Steinman RM. The dendritic cell system and its role in immunogenicity. Annu. Rev. Immunol. (1991) 9:271.[CrossRef][Web of Science][Medline]
  40. Jaksits S, Bauer W, Kriehuber E, et al. Lipid raft-associated GTPase signaling controls morphology and CD8+ T cell stimulatory capacity of human dendritic cells. J. Immunol. (2004) 173:1628.[Abstract/Free Full Text]
  41. Newton AC. Protein kinase C: structure, function, and regulation. J. Biol. Chem. (1995) 270:28495.[Free Full Text]
  42. Mellor H, Parker PJ. The extended protein kinase C superfamily. Biochem. J. (1998) 332(Pt 2):281.[Web of Science][Medline]
  43. Nishizuka Y. Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science (1992) 258:607.[Abstract/Free Full Text]
  44. Steinberg SF. Distinctive activation mechanisms and functions for protein kinase Cdelta. Biochem. J. (2004) 384:449.[CrossRef][Web of Science][Medline]
  45. Harder T, Scheiffele P, Verkade P, Simons K. Lipid domain structure of the plasma membrane revealed by patching of membrane components. J. Cell Biol. (1998) 141:929.[Abstract/Free Full Text]
  46. Nishizuka Y. Protein kinase C and lipid signaling for sustained cellular responses. FASEB J. (1995) 9:484.[Abstract]
  47. Villadangos JA, Cardoso M, Steptoe RJ, et al. MHC class II expression is regulated in dendritic cells independently of invariant chain degradation. Immunity (2001) 14:739.[CrossRef][Web of Science][Medline]
  48. Delamarre L, Holcombe H, Mellman I. Presentation of exogenous antigens on major histocompatibility complex (MHC) class I and MHC class II molecules is differentially regulated during dendritic cell maturation. J. Exp. Med. (2003) 198:111.[Abstract/Free Full Text]
  49. Holm PK, Eker P, Sandvig K, van Deurs B. Phorbol myristate acetate selectively stimulates apical endocytosis via protein kinase C in polarized MDCK cells. Exp. Cell Res. (1995) 217:157.[CrossRef][Web of Science][Medline]
  50. Cardone MH, Smith BL, Song W, Mochly-Rosen D, Mostov KE. Phorbol myristate acetate-mediated stimulation of transcytosis and apical recycling in MDCK cells. J. Cell Biol. (1994) 124:717.[Abstract/Free Full Text]
  51. Garcia-Garcia E, Rosales C. Signal transduction during Fc receptor-mediated phagocytosis. J. Leukoc. Biol. (2002) 72:1092.[Abstract/Free Full Text]
  52. Chimini G, Chavrier P. Function of Rho family proteins in actin dynamics during phagocytosis and engulfment. Nat. Cell Biol. (2000) 2:E191.[CrossRef][Web of Science][Medline]
  53. Garrett WS, Chen LM, Kroschewski R, et al. Developmental control of endocytosis in dendritic cells by Cdc42. Cell (2000) 102:325.[CrossRef][Web of Science][Medline]
  54. Gschwendt M, Muller HJ, Kielbassa K, et al. Rottlerin, a novel protein kinase inhibitor. Biochem. Biophys. Res. Commun. (1994) 199:93.[CrossRef][Web of Science][Medline]
  55. Kontny E, Kurowska M, Szczepanska K, Maslinski W. Rottlerin, a PKC isozyme-selective inhibitor, affects signaling events and cytokine production in human monocytes. J. Leukoc. Biol. (2000) 67:249.[Abstract]
  56. Davies SP, Reddy H, Caivano M, Cohen P. Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem. J. (2000) 351:95.[CrossRef][Web of Science][Medline]
  57. McKee AS, Dzierszinski F, Boes M, Roos DS, Pearce EJ. Functional inactivation of immature dendritic cells by the intracellular parasite Toxoplasma gondii. J. Immunol. (2004) 173:2632.[Abstract/Free Full Text]
  58. Haverstick DM, Engelhard VH, Gray LS. Three intracellular signals for cytotoxic T lymphocyte-mediated killing. Independent roles for protein kinase C, Ca2+ influx, and Ca2+ release from internal stores. J. Immunol. (1991) 146:3306.[Abstract]
  59. Pardo J, Buferne M, Martinez-Lorenzo MJ, et al. Differential implication of protein kinase C isoforms in cytotoxic T lymphocyte degranulation and TCR-induced Fas ligand expression. Int. Immunol. (2003) 15:1441.[Abstract/Free Full Text]
  60. Chen ZZ, Coggeshall KM, Cambier JC. Translocation of protein kinase C during membrane immunoglobulin-mediated transmembrane signaling in B lymphocytes. J. Immunol. (1986) 136:2300.[Abstract]
  61. DeFranco AL. Structure and function of the B cell antigen receptor. Annu. Rev. Cell Biol. (1993) 9:377.[CrossRef][Web of Science][Medline]
  62. Pleiman CM, D'Ambrosio D, Cambier JC. The B-cell antigen receptor complex: structure and signal transduction. Immunol. Today (1994) 15:393.[CrossRef][Web of Science][Medline]
  63. Barois N, Forquet F, Davoust J. Selective modulation of the major histocompatibility complex class II antigen presentation pathway following B cell receptor ligation and protein kinase C activation. J. Biol. Chem. (1997) 272:3641.[Abstract/Free Full Text]
  64. Anderson HA, Bergstralh DT, Kawamura T, Blauvelt A, Roche PA. Phosphorylation of the invariant chain by protein kinase C regulates MHC class II trafficking to antigen-processing compartments. J. Immunol. (1999) 163:5435.[Abstract/Free Full Text]
  65. Schechtman D, Mochly-Rosen D. Adaptor proteins in protein kinase C-mediated signal transduction. Oncogene (2001) 20:6339.[CrossRef][Web of Science][Medline]
  66. Caux C, Dezutter-Dambuyant C, Schmitt D, Banchereau J. GM-CSF and TNF-alpha cooperate in the generation of dendritic Langerhans cells. Nature (1992) 360:258.[CrossRef][Medline]
  67. Davis TA, Saini AA, Blair PJ, et al. Phorbol esters induce differentiation of human CD34+ hemopoietic progenitors to dendritic cells: evidence for protein kinase C-mediated signaling. J. Immunol. (1998) 160:3689.[Abstract/Free Full Text]
  68. St Louis DC, Woodcock JB, Franzoso G, et al. Evidence for distinct intracellular signaling pathways in CD34+ progenitor to dendritic cell differentiation from a human cell line model. J. Immunol. (1999) 162:3237.[Abstract/Free Full Text]
  69. Lindner I, Kharfan-Dabaja MA, Ayala E, et al. Induced dendritic cell differentiation of chronic myeloid leukemia blasts is associated with down-regulation of BCR-ABL. J. Immunol. (2003) 171:1780.[Abstract/Free Full Text]
  70. Cejas PJ, Carlson LM, Zhang J, et al. Protein kinase C betaII plays an essential role in dendritic cell differentiation and autoregulates its own expression. J. Biol. Chem. (2005) 280:28412.[Abstract/Free Full Text]
  71. Leitges M, Mayr M, Braun U, et al. Exacerbated vein graft arteriosclerosis in protein kinase Cdelta-null mice. J. Clin. Invest. (2001) 108:1505.[CrossRef][Web of Science][Medline]
  72. Liu H, Keefer JR, Wang QF, Friedman AD. Reciprocal effects of C/EBPalpha and PKCdelta on JunB expression and monocytic differentiation depend upon the C/EBPalpha basic region. Blood (2003) 101:3885.[Abstract/Free Full Text]
  73. Cejas PJ, Carlson LM, Kolonias D, et al. Regulation of RelB expression during the initiation of dendritic cell differentiation. Mol. Cell. Biol. (2005) 25:7900.[Abstract/Free Full Text]
  74. Bertho N, Blancheteau VM, Setterblad N, et al. MHC class II-mediated apoptosis of mature dendritic cells proceeds by activation of the protein kinase C-delta isoenzyme. Int. Immunol. (2002) 14:935.[Abstract/Free Full Text]
  75. Setterblad N, Roucard C, Bocaccio C, Abastado JP, Charron D, Mooney N. Composition of MHC class II-enriched lipid microdomains is modified during maturation of primary dendritic cells. J. Leukoc. Biol. (2003) 74:40.[Abstract/Free Full Text]
  76. Do Y, Hegde VL, Nagarkatti PS, Nagarkatti M. Bryostatin-1 enhances the maturation and antigen-presenting ability of murine and human dendritic cells. Cancer Res. (2004) 64:6756.[Abstract/Free Full Text]
  77. Schnare M, Barton GM, Holt AC, Takeda K, Akira S, Medzhitov R. Toll-like receptors control activation of adaptive immune responses. Nat. Immunol. (2001) 2:947.[CrossRef][Web of Science][Medline]
  78. West MA, Wallin RP, Matthews SP, et al. Enhanced dendritic cell antigen capture via toll-like receptor-induced actin remodeling. Science (2004) 305:1153.[Abstract/Free Full Text]
  79. Sallusto F, Cella M, Danieli C, Lanzavecchia A. Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: downregulation by cytokines and bacterial products. J. Exp. Med. (1995) 182:389.[Abstract/Free Full Text]
  80. Pierre P, Turley SJ, Gatti E, et al. Developmental regulation of MHC class II transport in mouse dendritic cells. Nature (1997) 388:787.[CrossRef][Medline]
  81. Kleijmeer MJ, Morkowski S, Griffith JM, Rudensky AY, Geuze HJ. Major histocompatibility complex class II compartments in human and mouse B lymphoblasts represent conventional endocytic compartments. J. Cell Biol. (1997) 139:639.[Abstract/Free Full Text]
  82. Kabir N, Schaefer AW, Nakhost A, Sossin WS, Forscher P. Protein kinase C activation promotes microtubule advance in neuronal growth cones by increasing average microtubule growth lifetimes. J. Cell Biol. (2001) 152:1033.[Abstract/Free Full Text]
  83. Vermassen E, Van Acker K, Annaert WG, et al. Microtubule-dependent redistribution of the type-1 inositol 1,4,5-trisphosphate receptor in A7r5 smooth muscle cells. J. Cell Sci. (2003) 116:1269.[Abstract/Free Full Text]
  84. Wubbolts R, Fernandez-Borja M, Oomen L, et al. Direct vesicular transport of MHC class II molecules from lysosomal structures to the cell surface. J. Cell Biol. (1996) 135:611.[Abstract/Free Full Text]
  85. Jordens I, Fernandez-Borja M, Marsman M, et al. The Rab7 effector protein RILP controls lysosomal transport by inducing the recruitment of dynein-dynactin motors. Curr. Biol. (2001) 11:1680.[CrossRef][Web of Science][Medline]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 J. Leukoc. Biol.Home page
C. Gelin, I. Sloma, D. Charron, and N. Mooney
Regulation of MHC II and CD1 antigen presentation: from ubiquity to security
J. Leukoc. Biol., February 1, 2009; 85(2): 215 - 224.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
19/6/719    most recent
dxm034v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Majewski, M.
Right arrow Articles by Boes, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Majewski, M.
Right arrow Articles by Boes, M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?