Differential regulation of maturation and apoptosis of human monocyte-derived dendritic cells mediated by MHC class II

Anna E. Lokshin1, Pawel Kalinski2, R. Rita Sassi1, Robbie B. Mailliard2, Jan Müller-Berghaus2, Walter J. Storkus2, Xiaojun Peng1, Adele M. Marrangoni1, Robert P. Edwards1 and Elieser Gorelik3

1 Department of Obstetrics/Gynecology and Reproductive Sciences, 2 Department of Surgery, and 3 Department of Pathology, University of Pittsburgh and University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213, USA

Correspondence to: A. Lokshin, Lab 320, MWRI, 204 Craft Avenue, Pittsburgh PA, 15213, USA. E-mail: lokshina{at}pitt.edu
Transmitting editor: D. Green


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Antigen-driven interaction of dendritic cells (DC) with CD4+ Th cells results in the exchange of bidirectional activating signals. Cross-linking of TCR by MHC class II-bound antigen activates Th cells, resulting in their up-regulation of CD40 ligand. Here we show that MHC class II molecules, in addition to their passive role in DC–Th cell interaction, can also actively induce DC maturation. Cross-linking of MHC class II molecules on human monocyte-derived DC results in the up-regulation of the surface expression of CD83, CD80, CD86, CD54, CD1a and CD40 molecules, the typical DC maturation-associated markers. It also promotes a rapid homotypic aggregation of DC paralleled by the up-regulation of such adhesion molecules as VLA-4, tissue transglutaminase, CD54 and CD11c. The impact of MHC class II cross-linking upon DC was context dependent. The outcome of MHC class II signaling depends on the maturation status of DC. While the cross-linking of MHC class II on immature DC promoted their maturation, the dominant effect upon the DC that were previously matured was the induction of DC apoptosis. Our current observations indicate that, in addition to the previously reported negative impact of MHC class II-mediated signaling on DC function, it also promotes DC maturation, participating in the enhancement of DC stimulatory function. Importantly, MHC class II-induced DC maturation and apoptosis are mediated by different signaling pathways, sensitive to different sets of inhibitors. This opens the possibility of differential regulation of each of these events in immunotherapy.

Keywords: apoptosis, dendritic cells, homotypic aggregation, maturation, MHC class II, signal transduction


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Dendritic cells (DC) are the most potent antigen-presenting cells (APC) and can play a critical role in the initiation of T cell-mediated immunity. Immature DC reside in most non-lymphoid tissues, and have high capability of antigen capture and processing. Following their activation, DC migrate to the draining lymphoid organs, where they mature and acquire the ability to present antigenic peptides to T lymphocytes [reviewed in (1,2)]. DC maturation is manifested by an increase in the expression of co-stimulatory molecules, such as CD80, CD86 and CD40, and a decreased capacity to process antigen. DC present antigenic peptides complexed with MHC class I and II molecules that are recognized by CD8+ or CD4+ T cells respectively (35).

The MHC class II molecules are constitutively expressed by the various APC, such as DC, macrophages and B cells, and have a crucial role in generating antigen-specific T cell responses. MHC class II are heterodimeric glycoproteins consisting of two non-covalently associated polymorphic chains, {alpha} (32–34 kDa) and ß (29–32 kDa). MHC class II chains consist of extracellular, transmembrane and cytoplasmic domains, and are expressed by the various APC, such as DC, macrophages and B cells [reviewed in (6)]. The extracellular portion of MHC class II molecules that consists of {alpha}1 and ß2 domains forms a groove containing nanomeric peptide that is recognized by TCR of CD4+ T cells. In addition, CD4 co-receptor binds the same MHC class II molecule (7,8). Engagement of TCR and CD4 molecules by MHC class II results in a well-documented signal transduction cascade inside CD4+ T cells leading to T cell activation [reviewed in (9)]. To verify whether the TCR–MHC class II interaction may result in a signal transduction also into APC, several studies were performed using antibody-mediated cross-linking of MHC class II molecules on B lymphocytes as a model. Cross-linking of MHC class II molecules by specific antibodies leads to either proliferation or apoptosis of B cells (10,11). These two outcomes were regulated by two separate MHC class II-initiated pathways, with proliferation requiring new gene transcription and activation of Src family tyrosine kinases, and apoptosis depending on serine/threonine phosphatases, and cytoskeletal mobility (1017). Activation and translocation of isoenzymes of the protein kinase C (PKC) family and intracellular calcium were shared between the two pathways (1017). Furthermore, MHC class II was shown to mediate aggregation of B lymphocytes via a protein tyrosine kinase (PTK)-dependent pathway that preceded activation of PKC and involved the LFA-1 molecules (13,14,18,19). The intracytoplasmic region of the HLA-DR chain was requisite for the principal signaling pathway initiated via MHC class II (6,16). These results imply that MHC class II molecules may function simultaneously both as ligands and receptors which send signals into the cells expressing these molecules.

Recently, several groups reported the induction of apoptosis after MHC class II ligation on human monocytes and DC (2024). According to these reports, in these cells, MHC class II cross-linking with specific antibodies led to caspase- and Fas-independent forms of apoptosis within 6–24 h (20,22). MHC class II ligation on human DC has been reported to stimulate tyrosine phosphorylation (25,26), suggesting the possibility of signal transduction from MHC class II molecules into DC. Recent data suggested that HLA-DR, -DQ and -DP molecules transmit signals to monocytes via MAP kinases and lead to distinct monokine activation patterns, which may affect T cell responses in vivo (16). While it has been reported that MHC class II may participate in the regulation of the cytokine production by DC (27), its impact on DC maturation has not been addressed. Here we report that ligation of MHC class II by specific antibody transduces a maturation-inducing signal into DC, suggesting an additional role of MHC class II–TCR binding during the antigen-driven cognate interaction of Th cells and DC. Our data indicate that the induction of DC maturation and apoptosis are regulated by different signaling cascades, and preferentially affect DC at different stages of maturation.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Reagents
Mouse anti-HLA-DR,DP,DQ mAb (Tü39, mouse IgG2a, {kappa}), anti-CD45 mAb, isotype-matched mouse IgG1 and all fluorochrome-conjugated antibodies for flow cytometry were purchased from PharMingen (San Diego CA). A series of 15 anti-MHC class II antibodies with different specificities was obtained from Terra Nova Biotechnology (St John’s, Newfoundland, Canada). Neutralizing anti-Fas mAb ZB4, polyclonal rabbit antibodies to Akt and its phosphorylated form (Ser473 pAkt) were from Cell Signaling (Beverly, MA). Monoclonal anti-Lyn, -Src, -Bcl-2, phosphotyrosine (PY99), as well as rabbit anti-Bax, and goat anti-caspase-3 and anti-{gamma}-actin antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). PKC inhibitor, H-7, mitogen-activated protein kinase kinase (MEK) inhibitor, PD 98059, and phosphatidylinositol (PI)-3 kinase inhibitor, wortmannin, were from Biomol (Plymouth Meeting, PA). Caspase inhibitor Z-Val- Ala-Asp(OMe)-CH2F (zVAD-fmk) was obtained from BD PharMingen (San Diego, CA) and zVAD–FITC was purchased from Promega (Madison, WI). Mitochondrial dye, DiOC6, was from Molecular Bioprobes (Eugene, OR).

Generation and culture of DC
Peripheral blood mononuclear cells (PBMC) were obtained from venous blood drawn from normal healthy volunteers at the Pittsburgh Central Blood Bank. PBMC were isolated by centrifugation on a Ficoll-Diatrizoate density gradient (ICN, Aurora, OH). Subsequently, the cells were separated on a Percoll (Sigma) gradient (1.076, 1.059 and 1.045 g/ml). Monocytes were further purified by a 45-min adherence step. Non-adherent cells were harvested, and monocytes (5 x 105 cells/ml) were cultured for 6 days in 24-well plates (Costar, Cambridge, MA) in IMDM with 10% FCS (Hyclone, Logan, UT) supplemented with granulocyte macrophage colony stimulating factor (GM-CSF; 1000 U/ml) (gift from Immunex, Seattle, WA) and IL-4 (800 U/ml) (Genzyme, Boston, MA). At day 6, the cultures consisted of uniformly HLA-DR+, CD83 and CD40high immature DC, without detectable CD3+ cells.

Treatment procedures
The viability of the cells preceding treatment was confirmed to be >90% by Trypan blue exclusion. DC were treated with 500 ng/ml of anti-MHC class II antibody, isotype-matched control IgG1 or anti-CD45 antibody for the indicated time periods. Based on the results of preliminary dose–response experiments (data not shown), this concentration of anti-MHC class II mAb was chosen as optimally stimulating. When the effects of different inhibitors were evaluated, drugs were added to the cells 1 h before anti-MHC or control antibodies.

Flow cytometry
Cells were pelleted and washed with FACS buffer (PBS, 1% BSA and 0.1% sodium azide). Cells were incubated with optimal concentrations of FITC- or phycoerythrin-conjugated primary antibodies for 30 min at 4°C. For all experiments, isotype control IgG1 or irrelevant control anti-CD45 mAb were included. Cells were then washed, fixed with 2% paraformaldehyde, and flow cytometric analysis was performed using a FACScan apparatus and CellQuest software from Becton Dickinson Immunocytometry Systems (San Jose, CA).

Cell death assays
Immature DC were stimulated with a cytokine maturation cocktail (MC) consisting of tumor necrosis factor (TNF)-{alpha} (10 ng/ml; Sigma, St Louis, MO), rhIL-1ß (10 ng/ml; Genzyme), rhIL-6 (1000 IU/ml; Novartis, Basel, Switzerland) and prostaglandin E2 (1 µg/ml; Sigma). Immature and mature DC were incubated with 500 ng/ml of anti-MHC class II antibody or IgG1 control in the presence or absence of inhibitors for 8 h. Apoptosis was evaluated using the Annexin V binding assay. Cells (5 x 105) were washed in PBS and 100 µl of FITC-conjugated Annexin V (5 µg/ml) in a calcium-containing buffer were added according to the manufacturer’s instructions (Clontech, Palo Alto, CA). After incubation for 10 min at room temperature, 400 µl of calcium-containing buffer was added and the samples were immediately analyzed by flow cytometry as described above. To assess loss of mitochondrial potential, DC were stained with 40 nM mitochondrial potential-sensitive dye DiOC6 for 15 min at 37°C according to the manufacturer’s protocol and changes of DiOC6 staining were analyzed by flow cytometry. When caspase inhibitor, zVAD-fmk, was used, DC were preincubated with 50 µM of this compound 1 h before addition of antibodies. After 4 h of incubation, more zVAD-fmk was added to a final concentration of 100 µM and incubation continued for a further 4 h.

Caspase activity assay
Total caspase activity was measured in DC treated with or without anti-MHC class II mAb using FITC–VAD-fmk caspase substrate (Promega) according to manufacturer’s instructions. Activated caspases covalently bind this substrate allowing for flow cytometric detection. DC were incubated with 10 nM FITC–VAD-fmk for 20 min at 37°C, washed with PBS and subjected to flow cytometric analysis.

Western blot analysis and immunoprecipitation
Lysates of DC (106 cells/sample) treated with anti-MHC class II or control IgG1 were prepared in a lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 4 mM EDTA, 1% Triton X-100, 20 µg/ml aprotinin, 10 µM leupeptin and 10 µM pepstatin A). To deplete lysates from anti-MHC class II IgG, they were further incubated with 20 µl of Protein A/G–agarose beads (Calbiochem) for 1 h at 4°C with shaking. Total cell extracts containing 100 µg total protein were separated on 12% SDS–PAGE, transferred to nitrocellulose membranes (Hybond-C; Amersham) and immunoblotted with specific antibodies as indicated. Proteins were visualized by enhanced chemiluminescence using Western blotting Luminol reagent (Santa Cruz Biotechnology).

Immunoprecipitation and immune complex kinase assay
Lyn and Src proteins were immunoprecipitated from total cell lysates. Immunoprecipitations were conducted for 1 h at 4°C using specific antibodies immobilized on Protein A/G–agarose beads (Santa Cruz). Immunoprecipitates were incubated for 20 min at 24°C with exogenous substrates in protein kinase buffer (50 mM HEPES, pH 7.4, 10 mM MgCl2, 10 mM MnCl2, 50 µM ATP and 250 µCi/ml [{gamma}-32P]ATP; 4500 Ci/mmol). [{gamma}-32P]ATP incorporation was analyzed by SDS–PAGE and autoradiography.

Cytokine release
DC were grown in 24-well plates at 1 x 106 cells/well and treated with or without anti-MHC class II antibody, isotype control IgG1 or MC for 24–48 h at 37°C. Following treat ment supernatants were collected and stored at –80°C. IL-6, IL-12p70, IFN-{gamma} and TNF-{alpha} concentrations were measured using ELISA kits (R & D Systems, Minneapolis, MN) according to the manufacturer-provided protocols. The detection limits for the kits were: IL-1ß (>30 pg/ml), TNF-{alpha} (>50 pg/ml), IL-6 (>50 pg/ml) and IL-12 p70 (>10 pg/ml).

Statistics
For each experiment DC from at least five donors were evaluated. All experiments were performed in triplicate, unless otherwise indicated, and mean values ± SD are presented. Comparisons between the values were performed using a two-tailed Student’s t-test. A value of P < 0.05 was considered statistically significant.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Effect of MHC class II cross-linking on DC adhesion
Immature DC were generated by culturing peripheral blood monocytes with GM-CSF and IL-4 for 6 days. These cells had typical DC morphology and were characterized by the absence of monocyte marker, CD14, and lineage-related antigens, CD3, CD56, CD19 and CD16 (not shown). They expressed CD80 (B7-1), CD86 (B7-2) and CD1a (Fig. 1), as well as MHC class I and II molecules (data not shown), all molecules that are involved in antigen presentation and T cell activation by APC (1,2). However, these DC expressed very low amounts of CD83, which is a marker of mature DC, and could, therefore, be characterized as immature (Fig. 1). To examine the direct effects of MHC class II triggering on DC, day 6 immature DC were exposed to 500 ng/ml of Tü39 anti-MHC class II mAb (for various time intervals. The most noticeable and immediate response of DC was a rapid (after 3–4 h) and robust cluster formation (Fig. 2A), indicating an increase in homotypic aggregation. No unclustered cells could be observed in anti-MHC class II antibody-treated DC cultures following their resuspension by gentle pipetting. In contrast, IgG1 or anti-CD45 antibody-treated DC cultures had 92.2 ± 14.3 and 93.0 ± 11.5% of non-clustered cells respectively. In order to investigate mechanisms of MHC class II-mediated homotypic aggregation of DC we analyzed the expression of adhesion molecules that are involved in cell–cell and cell–substrate binding of DC, such as CD11c (integrin {alpha}x chain) (28), CD54 (ICAM-1) (29,30) and VLA-4 ({alpha}4ß1 integrin) (31,32). Flow cytometric analysis revealed that anti-MHC class II antibody-treated DC had a higher surface expression of all three adhesion molecules as compared to untreated or anti-CD45 mAb-treated DC. Up-regulation of VLA-4 was most profound (Fig. 2B). Tissue transglutaminase (tTG) serves as a ligand for VLA-4 in non-hematopoietic cells, promoting cell adhesion and spreading (33,34). In our experiments, MHC class II-mediated homotypic clustering of DC was accompanied by a substantial increase in surface tTG expression (Fig. 2B).



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Fig. 1. Stimulation of DC maturation by anti-MHC class II mAb. Monocyte-derived DC were cultured with Tü39 antibody (MHC class II) or mouse control isotype IgG2a (Control) (each at 500 ng/ml) for 24 h. Expression of CD86, CD80, CD83 and CD1a was analyzed by flow cytometry using FITC- or phycoerythrin-conjugated mAb. Cells stained with FITC- or phycoerythrin-conjugated mouse IgG (IgG) served as control for non-specific antibody binding. Similar results were obtained in five independent experiments.

 


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Fig. 2. Anti-MHC class II mAb enhances adhesive properties of human DC. DC were incubated with 500 ng/ml of Tü39 antibody (MHC class II) or mouse IgG2a (Control). (A) Homotypic clustering was detected after 3 h of incubation. (B) Expression of adhesion-related molecules (VLA-4, tTG, CD54 and CD11c) was analyzed by flow cytometry after 24 h of incubation.

 
Effect of MHC class II cross-linking on expression of DC surface maturation markers
Homotypic clustering of DC closely correlates with their state of maturation (35). However, to this end, the effects of MHC class II cross-linking on DC maturation have not been examined. To test the impact of MHC class II triggering on expression of DC maturation markers, day 6 immature DC were exposed to 500 ng/ml of Tü39 for 24 h or to control isotype IgG1. Treatment of DC with Tü39 antibody resulted in up-regulation of CD83, CD80, CD86 and CD1a as compared to control (Fig. 1). Prolonged (up to 4 days) exposure of DC to anti-MHC class II antibody further potentiated maturation effects (data not shown). It is possible that just cross-linking of any molecules expressed on DC surface would non-specifically up-regulate maturation markers. To address this concern, we incubated DC with anti-CD45 antibody, which is abundant on DC. However, engagement of CD45 did not result in DC maturation (see below in Fig. 4).

To investigate whether anti-MHC class II mAb stimulated cytokine production by DC, we measured the concentration of proinflammatory cytokines, IL-1ß, TNF-{alpha} and IL-6, as well as IL-12 p70, in cell culture medium in the presence or absence of Tü39 or MC. Tü39 did not up-regulate the secretion of any of these cytokines (data not shown). It is of note that incubation of DC with MC also did not stimulate cytokine production (data not shown)

Engagement of MHC class II on DC triggers apoptotic cell death
MHC class II-mediated apoptosis in DC has been well documented (2024). Before we explored the relationship between maturation and apoptosis, we first re-evaluated MHC class II-mediated apoptosis in our experimental system. In agreement with these data, in our experiments the percentage of apoptotic DC increased from 10.6 ± 3.45% in IgG1-treated cells to 41.1 ± 13.41% in immature DC and from 14.5 ± 6.92 to 72.8 ± 17.12% in mature DC treated with Tü39 (Fig. 3). No apoptotic changes could be detected in anti-CD45 antibody or isotype-matched mouse IgG-treated DC (Fig. 4A). In agreement with published observations, neutralizing anti-Fas antibody, ZB4, did not abrogate MHC class II-induced apoptosis (Fig. 4A), suggesting that MHC class II-induced apoptosis is Fas–Fas ligand independent. However, contrary to the published observations (20,22,24), in our experimental system, the broad-spectrum caspase inhibitor/pseudosubstrate zVAD-fmk substantially inhibited MHC class II-induced apoptosis in DC (Fig. 4A), indicating that activation of caspases is required for MHC class II-induced cell death in DC. When DC were loaded with FITC-conjugated zVAD-fmk, MHC class II ligation resulted in increased intracellular fluorescence indicative of caspase activation (Fig. 5A). To ascertain the input of individual caspases in total caspase activity, Western blot analysis was performed using specific antibodies against caspase-3, -8, -9, and -10, known to be activated during different stages of apoptotic pathways (3638). Caspase-9 is the initiating caspase in mitochondrial apoptotic pathway that is death receptor independent (39,40). Once activated, caspase-9 can initiate a caspase cascade involving the downstream executioners caspase-3, -6 and -7 (40). Caspase-10 is a novel caspase homologous to caspase-8 whose physiological role is yet unknown (41). It has been demonstrated that caspase-10 mutations result in abnormal apoptosis of DC underlying a unique disorder of DC homeostasis (38). Western blot demonstrated cleavage of caspase-3, -9 and -10, but not of caspase-8, in response to stimulation with Tü39 (Fig. 5B).



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Fig. 3. Increased sensitivity of mature DC to apoptosis following MHC class II cross-linking. Immature and mature human monocyte-derived DC were treated with 500 ng/ml of Tü39 antibody for 8 h. DC were stained with Annexin V–FITC and analyzed by flow cytometry. The results of one of five representative experiments are presented.

 


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Fig. 4. Regulation of MHC class II-mediated maturation and apoptosis in human DC. Human monocyte-derived DC were treated with 500 ng/ml of anti-MHC class II mAb in the presence or absence of inhibitors (see Methods). DC incubated with 500 ng/ml of mouse IgG or anti-CD45 mAb served as controls. (A) Apoptosis measured as percent of Annexin V+ cells. (B and C) Expression of maturation markers CD83 and CD86 presented as MFI after subtraction of non-specific antibody binding. Each point presents the mean of three representative experiments. *P > 0.95.

 


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Fig. 5. Effect of anti-MHC class II antibody on caspase activity and mitochondrial membrane potential. Immature and mature DC were treated with isotype control IgG (Control) or anti-MHC class II mAb for 6 h (A and C) or for indicated time intervals (B). (A) The level of caspase activity was analyzed using the zVAD–FITC substrate by flow cytometry. (B) Expression of caspases was analyzed by Western blot using specific antibodies. Jurkat cells treated with 50 ng/ml of anti-Fas antibody for 6 h were used as a positive control for caspase-8 cleavage. (C) Mitochondria permeability transition was assessed by DiOC6 staining and analyzed by flow cytometry.

 
It has been documented that MHC class II ligation in mouse splenic DC results in mitochondrial permeability transition, indicating that MHC class II-mediated apoptosis in DC utilized the mitochondrial pathway (22). In agreement with these observations in our experiments, MHC class II cross-linking resulted in pronounced loss of mitochondrial dye DiOC6 staining, indicating altered mitochondrial permeability transition (Fig. 5C). However, in contrast to the cited publication, bongkrekic acid, which is an inhibitor of the mitochondrial permeability transition, reduced apoptosis as measured by Annexin V binding (Fig. 4A) in human monocyte-derived DC, implicating the mitochondrial pathway in the MHC class II-mediated apoptotic cell death of human DC. We next examined the effects of MHC class II cross-linking on expression of anti-apoptotic protein, Bcl-2, and pro-apoptotic protein, Bax, in human DC. Interestingly, increased expression of both proteins was observed following 24 h of anti-MHC class II antibody treatment (Fig. 6). In addition, Tü39 up-regulated the expression of p53 protein in human DC (Fig. 6).



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Fig. 6. Anti-MHC class II antibody up-regulates the expression of apoptosis-regulating proteins. Mature DC were treated with anti-MHC class II mAb for the indicated time periods. Expression levels of Bcl-2, Bax and p53 were analyzed by Western blot analysis with specific antibodies. Expression of {gamma}-actin serves as a loading control.

 
MHC class II-induced maturation and apoptosis of human DC are regulated via different signaling pathways
We next proceeded to ascertain the regulation of maturation and apoptosis of human DC following MHC class II cross-linking. To accomplish this task, the specific inhibitors of several signal transduction pathways were employed. DC were pretreated with either protein kinase A (PKA)/PKC inhibitor, H-7, broad spectrum tyrosine kinase inhibitor, genistein, PI-3 kinase inhibitor, wortmannin or MEK kinase inhibitor, PD 98059, for 1 h before stimulating with anti-MHC class II. The selected concentrations of inhibitors have been demonstrated to inhibit corresponding enzymes in DC or monocytes (12,42). At selected concentrations, neither inhibitor alone affected the viability of DC (data not shown). We evaluated the effect of kinase inhibitors on MHC class II-mediated apoptosis. MHC class II-induced apoptosis depended on the MEK kinase/ERK pathway, since it was completely inhibited by PD 98059 (Fig. 4A). Additionally, PI-3 kinase inhibitor, worthmannin, substantially inhibited MHC class II-induced apoptosis in DC (Fig. 4A). We further examined the effect of these inhibitors on the MHC class II-induced maturation of DC by testing the expression of maturation-associated molecules, CD83, CD80, CD86, CD11c and CD40. We have observed that MHC class II-mediated up-regulation of different maturation-associated proteins was inhibited by a distinct set of inhibitors. However, contrary to Annexin V binding, the up-regulation of all these markers required tyrosine phosphorylation since it was completely abrogated by genistein (Fig. 4B and C, and data not shown). We chose to present here the effect of the above inhibitors on the expression of two representative markers, CD86 and CD83 (Fig. 4B and C). As shown, in addition to the requirement for tyrosine phosphorylation, MHC class II-driven up-regulation of CD86 depended on the PKA or PKC pathway since it was inhibited by H-7. However, the above pathway was not involved in MHC class II-mediated up-regulation of CD83. It is possible that the apoptosis is the primary effect of MHC class II cross-linking resulting in phagocytosis of dead DC by bystander DC, which may lead to their maturation. To test this hypothesis, we examined the effect of MHC class II cross-linking by Tü39 antibody in the presence of zVAD-fmk and bongkrekic acid, both of which efficiently inhibit MHC class II-mediated apoptosis in DC. Neither inhibitor affected MHC class II-mediated maturation of DC (Fig. 4B and C).

Effect of different maturation agents and various anti-MHC class II antibodies on maturation and apoptosis of human DC
To investigate whether maturation of DC is always paralleled with apoptosis, we examined Annexin V binding in DC following incubation with the agents, known to be potent maturation inducers, lipopolysaccharide (LPS) and TNF-{alpha} and with a cytokine MC consisting of TNF-{alpha} (10 ng/ml), rhIL-1ß (10 ng/ml), rhIL-6 (1000 IU/ml) and PGE2 (1 µg/ml) (3). As shown in Table 1, stimulation of DC with LPS, but not with TNF-{alpha} and MC, was accompanied by apoptosis. To further examine the effects of MHC class II cross-linking on DC maturation and apoptosis, DC were incubated with a panel of 16 anti-MHC class II mAb with different epitope specificities for 24 h and tested for expression of CD86. As shown in Table 1, all anti-HLA-DQ and -DR antibodies tested up-regulated CD86 regardless of their epitope specificity. However, three out of eight anti-HLA-DP mAb did not affect the expression of this maturation marker. Next, DC were incubated with these antibodies for 8 h and tested for apoptosis by Annexin V binding. All examined anti-HLA-DR antibodies induced apoptosis. However, one anti-HLA-DP and one of the two anti-HLA-DQ mAb failed to induce apoptosis in human DC (Table 1).


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Table 1. Effects of different maturation agents and anti-MHC class II antibodies on apoptosis and maturation of human DC
 
MHC class II ligation drives tyrosine phosphorylation and activation of Akt kinase
We next proceeded to investigate whether MHC class II ligation triggers signal transduction cascade in human DC. First the changes in protein tyrosine phosphorylation in anti-MHC class II antibody-treated DC versus control DC were examined by Western blot analysis using specific anti-phosphotyrosine antibody, PY99. MHC class II cross-linking consistently induced tyrosine phosphorylation of three proteins with apparent mol. wt ~60, 32 and 25 kDa (Fig. 7). Tyrosine phosphorylation of these proteins was completely prevented by pre-incubation of DC with the PTK inhibitor, genistein (data not shown). Given that one of the phosphorylated proteins detected in our assay has mol. wt of ~60 kDa, it may correspond to activated p53/56lyn or p60Src kinases (25,43,44). However, the immunoprecipitation of these kinases followed by immune complex kinase assay did not reveal their activation following anti-MHC class I treatment of DC (Fig. 7). Therefore, the nature of proteins that become phosphorylated upon MHC class I cross-linking remains unknown and may deserve further investigation.



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Fig. 7. MHC class II cross-linking induces protein tyrosine phosphorylation. DC (106 cells/sample) were treated with or without anti-MHC class I mAb for indicated time intervals at 37°C and then subjected to Western blot analysis. (A) MHC class II cross-linking induces tyrosine phosphorylation of 60-, 32- and 25-kDa proteins (arrows) as determined by staining with anti-phosphotyrosine (PY99) mAb. (B) Activation of Lyn and Src kinases was assayed by immune complex kinase assay and autoradiography. Equal loading of Src and Lyn proteins was confirmed by staining with appropriate antibodies (not shown). Activation of Akt was evaluated using phospho-Akt (Ser473) or pan-Akt antibodies.

 
We next explored the possibility that engagement of MHC class I on human DC could activate serine-threonine kinase activity. MHC class II cross-linking on human DC did not stimulate the activity of PKC and PKA kinases (data not shown). However, anti-MHC class II antibody-treated DC displayed a higher degree of phosphorylation and hence activation of Akt/protein kinase B, a protein kinase that is downstream of PI-3 kinase (Fig. 7).


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Data presented in this study indicate that the ligation of MHC class II molecules induces a signaling cascade in human monocyte-derived DC, and modulates their maturation status, stimulatory function and survival. To the best of our knowledge, this is the first discovery of MHC class II-mediated induction of maturation in DC.

Cross-linking of MHC class II molecules elicited several measurable effects. We observed that anti-MHC class II mAb potently stimulated homotypic aggregation of DC. Homotypic aggregation of DC may correlate with their T cell stimulating capabilities, as has been demonstrated in syngeneic mixed lymphocyte reactions (35). Enhanced homotypic aggregation may result from MHC class II-mediated up-regulation of CD54 (ICAM-1), CD11c (integrin {alpha}x chain) and especially VLA-4 (CD49d) molecules that determine adhesive properties of DC (28,29,45,46). VLA-4 directly interacts with multiple cell surface proteins on hematopoetic and non-hematopoietic cells, such as fibronectin and VCAM-1. It has been shown that the {alpha}4 subunit (CD49d) could also serve as a ligand for VLA-4 itself (47). Thus cells expressing VLA-4 could interact with each other and form aggregates via VLA-4 molecules. Cross-linking enzyme tTG represents another ligand for VLA-4. tTG plays an important role in adhesion and migration of monocytic cells (48), mediating VLA-4 association with fibronectin and potentiating integrin-mediated signaling (33,49). Induction of tTG expression upon MHC class II cross-linking suggests that tTG may play a role in MHC class II-mediated homotypic aggregation of human DC.

Ligation of MHC class II by specific mAb leads to up-regulation of several cell surface molecules including co-stimulatory molecules, CD80 and CD86, that are actively involved in antigen presentation and T cell activation (1,2). These phenotypic changes are typical for DC maturation, which is further confirmed by induction of expression of CD83, which is a marker of mature DC (1,2). Interestingly, MHC class II cross-linking does not induce the synthesis of DC proinflammatory cytokines, such as IL-1ß, TNF-{alpha} and IL-6, indicating that the MHC class II-induced DC maturation is a direct phenomenon and not a secondary effect of the induction of maturation-inducing cytokines. MHC class II-induced DC maturation occurs in the absence of the induction of IL-12p70. Such a ‘silent’ pattern of DC maturation is also induced by the inflammatory cytokine TNF-{alpha}, alone or in combination with another inflammatory mediator, prostaglandin E2 (50). In contrast, factors such as CD40 ligand, TRANCE, LPS, SAC and other bacterial products, induce an inflammatory pattern of DC maturation, associated with the production of IL-12p70 and other DC-derived cytokines (5155).

In accordance with previous publications (2024), cross-linking of MHC class II molecules on human DC increases binding of Annexin V to the DC cell surface. It has been observed that DC undergo apoptosis upon interaction with antigen-specific T lymphocytes (4,56). MHC class II-mediated apoptosis could be accountable for their death after accomplishing their antigen-presenting function to CD4+ T lymphocytes. The current observations that mature DC are more prone to undergo spontaneous apoptosis and that an additional activating/maturation-inducing signal, such as MHC class II-triggering, induces their death suggest that the optimal immunogenic activity of DC is limited to a relatively narrow time window following their activation.

Several studies reported the caspase independence of MHC class II-mediated apoptosis in DC based on the lack of inhibition of HLA-DR-mediated apoptosis by zVAD-fmk or zDEVD-fmk (20,22). In our hands, however, zVAD-fmk was able to inhibit MHC class II-mediated apoptosis. The difference in the results obtained may be explained by different schedules of zVAD-fmk administration. In our experiments, we pre-incubated cells for 30 min before addition of the Tü39 antibody and then added more inhibitor 4 h later. Our rationale was that the reported half-life for irreversible caspase inhibition by zVAD-fmk in vitro is <=40 min. In agreement with the demonstrated role of caspase activation in MHC class II-mediated apoptosis in DC, we have observed activation of caspase-3, -9 and -10, but not caspase-8.

Inhibition of apoptosis by bongkrekic acid, which is an inhibitor of the mitochondrial permeability transition (57), implicates the mitochondrial pathway in MHC class II-mediated apoptosis. Our experiments revealed depolarization of mitochondrial membranes following MHC class II cross-linking. Furthermore, anti-MHC class II antibody activated caspase-9, which is the initiating caspase in the mitochondrial apoptotic pathway that is death receptor independent (39,40). Engagement of MHC class II up-regulated two apoptosis-related proteins, Bcl-2 and Bax, that are localized to the mitochondrial membrane [for review, see (58)]. Furthermore, we have detected the up-regulation of expression of tumor suppressor gene, p53. p53 mediates apoptosis through a linear pathway involving up-regulation of Bax, release of cytochrome c from mitochondria and activation of caspases (59,60). Therefore, it is possible that MHC class II cross-linking up-regulates p53, which in turn initiates the mitochondrial cascade. These observations further signify the role of mitochondrial pathway in MHC class II-mediated apoptosis in DC.

Anti-MHC class II-induced increase in anti-apoptotic protein, Bcl-2, is somewhat surprising. Since no apoptotic cells remain in culture after 24 h, probably due to phagocytosis by surviving DC [(21) and our unpublished observation], it is possible that only the cells that express sufficient levels of Bcl-2 to protect them from the apoptotic effects of Bax were able to survive. On the other hand, increased expression of Bax may be responsible for higher sensitivity of mature DC to induction of apoptosis. Both Fas–Fas ligand independence and involvement of mitochondria imply that MHC class II-mediated apoptosis occurs via the mitochondrial rather than via death receptor signaling pathway (3638,61). CD95 ligand and MHC class II ligation are likely to present distinct pathways for the elimination of DC at different stages of maturation (22).

The results of our study demonstrate the differential regulation of MHC class II-mediated maturation and apoptosis in human DC. Apoptosis is not required for MHC class II-mediated maturation of DC. Furthermore, MC TNF-{alpha} and some anti-MHC class II antibodies that induce apoptosis do not affect maturation and vice versa. Finally, different signal transduction pathways regulate these two processes. Tyrosine phosphorylation appears to be critical for MHC class II-mediated maturation, but not for apoptosis. Interestingly, the pattern of tyrosine phosphorylation following cross-linking of class II molecules on human Langerhans cells was shown to be similar to that induced by contact sensitizers that are known to activate these cells (25). On the contrary, MHC class II-induced apoptosis, but not maturation, depends on MEK kinase/ERK and PI-3 kinase pathways. Our data agree with published evidence that different signaling pathways regulate maturation and apoptosis in LPS-stimulated human DC. MEK kinase/ERK and PI-3 kinase pathways are essential for survival of LPS-stimulated DC (42,62), whereas NF-{kappa}B is responsible for DC maturation (42).

Collectively, our results demonstrate that ligation of MHC class II molecules on DC can trigger both maturation and apoptosis of DC. The demonstration that the MHC class II-induced DC maturation and apoptosis are sensitive to different sets of inhibitors and preferentially affect different DC populations suggests the possibility of modulating of the balance between these events in immunotherapy.


    Acknowledgements
 
This work was supported by Magee-Womens Health Foundation Award (A. E. L.).


    Abbreviations
 
APC—antigen-presenting cell

DC—dendritic cells

GM-CSF—granulocyte macrophage colony stimulating factor

LPS—lipopolysaccharide

MEK—mitogen-activated protein kinase

MC—maturation cocktail

PBMC—peripheral blood mononuclear cell

PI-3-kinase phosphotidylinositol-3 kinase

PKA—protein kinase A

PKC—protein kinase C

PTK—protein tyrosine kinase

tTG—tissue transglutaminase

TNF—tumor necrosis factor


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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