The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yu, Q.
Right arrow Articles by Ostrowski, M. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yu, Q.
Right arrow Articles by Ostrowski, M. A.
The Journal of Immunology, 2004, 172: 6047-6056.
Copyright © 2004 by The American Association of Immunologists

The Role of the p38 Mitogen-Activated Protein Kinase, Extracellular Signal-Regulated Kinase, and Phosphoinositide-3-OH Kinase Signal Transduction Pathways in CD40 Ligand-Induced Dendritic Cell Activation and Expansion of Virus-Specific CD8+ T Cell Memory Responses 1

Qigui Yu*, Colin Kovacs{ddagger}, Feng Yun Yue* and Mario A. Ostrowski2,*,{dagger}

* Clinical Sciences Division and {dagger} St. Michael’s Hospital, University of Toronto, and {ddagger} Canadian Immunodeficiency Research Collaborative, Toronto, Ontario, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mature dendritic cells (DCs) are central to the development of optimal T cell immune responses. CD40 ligand (CD40L, CD154) is one of the most potent maturation stimuli for immature DCs. We studied the role of three signaling pathways, p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), and phosphoinositide-3-OH kinase (PI3K), in CD40L-induced monocyte-derived DC activation, survival, and expansion of virus-specific CD8+ T cell responses. p38 MAPK pathway was critical for CD40L-mediated up-regulation of CD83, a marker of DC maturation. CD40L-induced monocyte-derived DC IL-12 production was mediated by both the p38 MAPK and PI3K pathways. CD40L-mediated DC survival was mostly mediated by the PI3K pathway, with smaller contributions by p38 MAPK and ERK pathways. Finally, the p38 MAPK pathway was most important in mediating CD40L-stimulated DCs to induce strong allogeneic responses as well as expanding virus-specific memory CD8+ T cell responses. Thus, although the p38 MAPK, PI3K, and ERK pathways independently affect various parameters of DC maturation induced by CD40L, the p38 MAPK pathway within CD40L-conditioned DCs is the most important pathway to maximally elicit T cell immune responses. This pathway should be exploited in vivo to either completely suppress or enhance CD8+ T cell immune responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Strong T cell immune responses are instrumental in controlling a number of viral infections including EBV, CMV, and HIV-1 infection (1, 2, 3). Mature dendritic cells (DCs) 3 are central to the development of optimal T cell immune responses (4). A number of stimuli, including many of the TNF family member molecules, LPS, influenza infection, CpG DNA motifs, dsRNA, and pox viruses have been shown to facilitate the maturation of immature DCs resulting in their enhanced ability to present Ag to T cells and activate them (5, 6, 7, 8, 9, 10). Of these, we and others have shown that the TNF family molecule CD40 ligand (CD40L, CD154) is one of the most potent maturation stimuli for immature DCs (11, 12, 13, 14). CD40 ligation of immature DCs results in a cascade of morphologic and functional effects that include the following: 1) increased expression of the dendritic cell maturation marker CD83 and down-regulation of surface CD1a; 2) up-regulation of surface MHC class I and class II molecules, the costimulatory molecules CD80 (B7-1) and CD86 (B7-2), and the adhesion molecules CD54 (ICAM-1) and CD58 (LFA-30); 3) the production of proinflammatory cytokines IL-1, IL-6, and TNF-{alpha}; 4) the production of the T cell growth and differentiation factors, IL-12 and IL-15; and 5) enhanced DC survival (6, 11, 12, 14, 15, 16, 17, 18, 19, 20). Taken together, these effects have been shown to be important for optimally priming CD8+ T cell responses as well as for maintaining CD8+ T cell memory. CD154 is expressed predominantly on activated CD4+ T cells, and signaling through CD40 on DCs has been shown to be one of the primary mechanisms for CD4+ Th of CD8+ T cell responses (21, 22, 23, 24). Although some cellular immune responses can occur without CD154/CD40 interactions, greatly impaired CD4+ T cell as well as CD8+ T cell priming is often observed in the absence of this receptor/ligand interaction (25, 26, 27, 28).

The cellular signaling pathways associated with CD154/CD40 interactions have been elucidated mainly in studies with nonlymphoid cells or transformed B cell lines. Very few research groups have directly studied CD40 signaling pathways in DCs. Interestingly, CD40 itself lacks intrinsic kinase activity and thus mediates its intracellular function primarily through cytoplasmic tail-mediated recruitment of TNFR-associated factor (TRAF) signaling adaptors, a characteristic of members of the TNFR family (29, 30, 31). For CD40, TRAF2, 3, and 6 have been shown to bind its cytoplasmic tail. Because TRAFs themselves have no known enzyme activity they serve as adapter proteins for downstream signaling pathways. Engagement of CD40 on DCs has been shown to result in activation of the following signaling pathways: the Src family kinase, Lyn (32), NF-{kappa}B (33), the three members of the mitogen-activated protein kinase (MAPK) family including the extracellular signal-regulated kinases (ERKs), the c-jun N-terminal kinases (JNKs), and the p38 MAPK (34), as well as the phosphoinositide-3-OH kinase (PI3K) pathway (35).

The MAPKs have been shown to be involved in all aspects of the immune response, including the activation of innate and adaptive immune responses and regulation of apoptosis (36). Among the MAPKs, p38 MAPK and ERK have both been shown to induce the IL-12 p40 subunit in DCs, with p38 MAPK being responsible for the majority of IL-12 p40 induction (34, 37). IL-12 production by DC is critical for the differentiation of IFN-{gamma}-producing Th1 T lymphocytes (38). In a murine model, CD40-induced p38 MAPK and ERK activation were shown to be regulated upstream by TRAF6 (37). The p38 MAPK was also shown to up-regulate the antiapoptotic protein, cIAP2, which is the cellular inhibitor of apoptosis protein 2, thus implying a role for maintaining DC survival. In LPS-stimulated murine DC, ERK was also shown to be very important for DC survival after maturation (39).

Activation of NF-{kappa}B in DC has been shown to up-regulate the DC costimulatory molecules, CD80, CD86, and HLA-DR as well as the maturation marker CD83 but has very little effect on DC survival (39, 40).

The PI3K-Akt pathway has been shown to play a major role in many cell survival pathways (41). In murine DC, TRAF6 has been shown to phosphorylate c-src, which in turn phosphorylates PI3K. Activated PI3K converts plasma membrane lipid phosphatidylinositol-4,5-biphosphate to phosphatidylinositol-3,4,5-triphosphate. The serine-threonine kinase Akt is then recruited to the membrane and is phosphorylated by phosphoinositide-dependent kinase 1. In various cell types, activated Akt has been shown to promote cell survival and protect against apoptosis through multiple mechanisms, including suppressing caspase 3 and caspase 9 activation, promoting production of Bcl-xL, repressing the activity of the proapoptotic factor BAD, and suppressing the production of Fas ligand (41, 42, 43, 44, 45).

Thus, engagement of CD40 on DC results in the activation of a number of signaling pathways, with the end result being a mature activated DC that can optimally present Ag and activate responding T cells. (14, 21, 22, 31, 46, 47, 48). It is unclear what role these different signaling pathways within DCs have in the final immune response. Identifying the functional role of various distinct signaling pathways may be important for the design of effective adjuvants to enhance various aspects of the immune response or in the development of immunosuppressive agents for the treatment of organ transplantation. Using an in vitro T cell-DC coculture system, the current study examines the role of three signaling pathways, p38 MAPK, ERK, and PI3K, in CD40L (CD154)-induced monocyte-derived DC (MDDC) activation, survival, and expansion of viral-specific CD8+ T cell responses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Study subjects

Three HIV-1-uninfected individuals (participants 1–3), who were HLA-A*0201-positive and had detectable EBV-specific CD8+ T cell IFN-{gamma} responses by ELISPOT assay (data not shown), were recruited for leukopheresis to obtain large amounts of PBMCs. Three HIV-1-seropositive individuals were also studied (participants 4–6). This group included a long-term nonprogressor (participant 6), who was HIV-1-infected for 10 years, had stable CD4+ T cell counts >500/µl, and a viral load <50 copies/ml by bDNA, and two asymptomatic chronic progressors, the first of whom (participant 4) was HIV-1-infected for 5 years, CD4+ T cell count 410/µl, viral load 45,000 copies/ml, and the second (participant 5), who was HIV-1-infected for 5 years, CD4+ T cell count 700/µl, viral load 44,514 copies/ml. All HIV-1-infected individuals were not taking antiretroviral drugs during the study. Before the study, individuals were class I HLA typed and screened for EBV- or HIV-1-specific CTL by culturing PBMCs with HLA-restricted EBV or HIV-1 peptides and detecting IFN-{gamma}-producing CD8+ T cells by ELISPOT assay as previously described (14). Participants 1–3 responded to the HLA-A*0201-restricted BMLF1 region of EBV, (GLCLVAML); participant 4 demonstrated responses to an HLA-A24-restricted epitope (KYKLKHIVW) of HIV-1 p17 region of gag; participant 5 showed responses to the HLA-A*0201-restricted LMP-2 region of EBV (CLGGLLTMV); and participant 6 showed responses to an HLA-B27-restricted epitope (KRWIIGLNK) of HIV-1 p24 region of gag. Informed consent was obtained from participants in accordance with the guideline for conduction of clinical research at the University of Toronto and St. Michael’s Hospital (Toronto, Ontario, Canada). All investigational protocols were approved by the University of Toronto and St. Michael’s Hospital institutional review boards.

Abs and reagents

The following anti-human mAbs or polyclonal Abs conjugated with fluorochrome were purchased from BD PharMingen (San Diego, CA): Anti-CD3-allophycocyanin, Anti-CD3PerCP, anti-CD8FITC, anti-CD14FITC, anti-CD1aFITC, anti-CD80FITC, anti-CD83PE, anti-CD86FITC, anti-HLA-DR-allophycocyanin, anti-caspase-3FITC, anti-Bcl-2PE, and matched-isotype control Abs conjugated with FITC, PE, PerCP, or allophycocyanin. Annexin V-FLUOS Staining kit containing Annexin VFITC was purchased from Boehringer Mannheim (Roche Diagnostics, Indianapolis, IN). Anti-Akt Ab, anti-phospho-Akt Ab, anti-p38 MAPK Ab, anti-phospho-p38 MAPK Ab, anti-ERK1/2 Ab, and anti-phospho-ERK1/2 Ab were purchased from Cell Signaling Technology (Pickering, Ontario, Canada). PI3'K inhibitor LY294002, p38 MAPK inhibitor SB203580, and ERK inhibitor PD98059 were purchased from Sigma-Aldrich (Ontario, Canada) and dissolved in DMSO (Sigma-Aldrich). Recombinant human GM-CSF and IL-4 were purchased from ProTech (Rocky Hill, NJ). Trimeric CD40L was obtained as a gift from Immunex (Seattle, WA).

Generation of MDDCs

MDDCs were generated by a modification of a method previously described (9, 14). Briefly, PBMCs obtained by the Ficoll-Paque gradient centrifugation (Amersham Pharmacia Biotech, Uppsala, Sweden) were separated on multistep Percoll gradients (Sigma-Aldrich). The recovered monocytes were depleted of contaminating B cells, T cells, NK cells, and granulocytes using Ab-conjugated magnetic beads from the Monocyte Negative Isolation kit (Dynal Biotech, Oslo, Norway). Purified monocytes were cultured at 1 x 106 cells/ml in complete RPMI 1640 medium consisting of 10% FCS, 2 mM glutamine, 25 mM HEPES, and antibiotics in the presence of 50 ng/ml recombinant human GM-CSF and 100 ng/ml recombinant human IL-4. GM-CSF and IL-4 were added again on days 3 and 5 with the fresh complete RPMI 1640 medium. After 7 days of culture, over 50% of the cells were CD1a+, MHC class II+, CD80low, and CD14–, which represents an immature DC phenotype.

Allogeneic mixed lymphocyte proliferation

Primary MLRs were set up in 96-well round-bottom plates (Costar, Corning, NY), in a 0.2 ml of RPMI 1640 medium with 10% FCS in triplicate wells. PBMCs were used as responders and allogeneic MDDC were used as stimulators. The ratio of responder to stimulator (T cell to APC) was 20:1 for PBMCs and allogeneic MDDCs. The culture period was 3 days and cells were pulsed with [3H]thymidine (2 µCi/well; PerkinElmer Life Sciences, Boston, MA) for the last 6 h of culture. After harvesting cells, incorporated radioactivity was measured in an LS 6500 Multipurpose Scintillation Counter (Beckman Coulter Instruments, Fullerton, CA).

Induction of peptide-specific CTL

The protocol for expanding circulating memory CD8+ CTL ex vivo was previously described (14). Immature MDDCs that were stimulated for 3 days with trimeric CD40L, were pulsed with the specific HLA class I-restricted peptide at 40 µg/ml for 1 h at 37°C, then plated in 24-well plates (5 x 105 peptide pulsed or nonpeptide pulsed MDDCs/well) in RPMI 1640 medium supplemented with 10% FCS, 2 mM glutamine, 25 mM HEPES, and antibiotics. Freshly isolated or thawed autologous PBMCs were added to MDDCs at a ratio of 1:10 (5 x 106 cells/well in 2 ml medium). On day 3 and day 5, the medium was changed. No cytokines were added. On day 7, duplicate wells were pooled and cells were harvested and tested for CTL activity by intracellular IFN-{gamma} staining. Experiments were repeated in HIV-1-negative participant 1 and HIV-1-positive participant 3.

Flow cytometric analysis of cell surface and intracellular molecules

PBMCs or MDDCs were stained in PBS/1% FCS/0.02% NaN3 using fluorochrome-conjugated Abs obtained from BD PharMingen. Samples were performed using a FACSCaliber (BD Biosciences, San Diego, CA), and the data were analyzed using FlowJo software (Tree Star, San Carlos, CA). Appropriate isotype controls were used at the same protein concentration as the test Ab. For intracellular staining, cells were permeabilized using Cytofix/Cytoperm Plus kit (BD PharMingen) according to the manufacturer’s instructions. Intracellular staining was performed for detecting IL-12, caspase 3, and Bcl-2 using anti-human IL-12 (p40/p70, clone C11.5), anti-human active caspase-3FITC, and anti-human Bcl-2PE, respectively. Intracellular staining was also performed to enumerate the number of IFN-{gamma}-producing CD8+ T cells, as previously described (14). Briefly, for peptide-specific IFN-{gamma} staining, 0.25 x 106 cells were cultured in U-bottom 96-well plates in the presence of peptide-pulsed (~1–10 µM) autologous B lymphoblastoid cell lines or autologous T cell-depleted PBMCs as stimulator cells; nonpeptide-pulsed stimulator cells were used as background controls. Positive control cells were stimulated with the bacterial superantigen staphylococcal enterotoxin B (1 µg/ml) (Sigma-Aldrich). Cells were incubated with peptide-pulsed or nonpeptide-pulsed stimulator cells for 6 h at 37°C in 6% CO2. Monensin was added for the duration of the culture period to facilitate intracellular cytokine accumulation. For IFN-{gamma} staining, we used clone 4S.B3 to identify IFN-{gamma}-containing cells and clone MOPC-21 as the isotype control Ab (BD PharMingen).

Cell survival analysis and inhibitor treatment

Duplicate or triplicate wells containing 0.1 x 106 immature MDDCs per well were cultured for 48 h in round-bottom 96-well plates in complete RPMI 1640 medium in the presence or absence of 2 µg/ml trimeric CD40L. Cell apoptosis was assessed for the phosphatidylserine on the outer leaflet of apoptotic cell membranes using annexin V-fluorescein staining (Boehringer Mannheim) according to the manufacturer’s protocol. Cell apoptosis was also determined by detecting intracellular active caspase 3 using a FITC-conjugated-specific Ab for active caspase 3. Bcl-2 was also assessed by intracellular staining of MDDCs with PE-conjugated Bcl-2. For signaling kinase inhibitor experiments involving the PI3K inhibitor LY294002, the MAP/ERK inhibitor PD98059, or the p38 MAPK inhibitor SB203580, cells were preincubated with the indicated inhibitors for 30 min, then incubated with 2 µg/ml trimeric CD40L. DMSO was used as background control, as the inhibitors were dissolved in DMSO. Inhibitors were titrated at concentrations ranging from 5 to 50 mM. A dose of 25 mM for all inhibitors was used for all experiments as this was the lowest concentration that could completely inhibit kinase activity. Inhibitors were removed by twice washing MDDCs with 2%FCS/PBS before coculturing with T cells.

Western blot analysis

Immature MDDCs (5–10 x 106 cells) were incubated with 2 µg/ml trimeric CD40L or medium alone and harvested at indicated time points. Cells were washed once with ice-cold PBS, then resuspended in 100 µl of cell lysis buffer (Cell Signaling Technology). Cellular debris was removed by centrifugation and protein-containing supernatant was stored at –80°C until further use. Equal amounts (15 µl per lane) of the protein-containing supernatants were mixed with 5 µl of 4x NuPAGE SDS Sample buffer (Invitrogen, Carlsbad, CA) and boiled for 5 min, then subjected to NuPAGE Novex high-performance, precast gel (Invitrogen) and blotted onto polyvinylidene difluoride membranes (Millipore, Billerica, MA). Membranes were blocked with blocking buffer (5% nonfat dry milk in TBST buffer, (50 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20), pH 7.6) for 1 h at room temperature and incubated at 4°C overnight with primary Ab specific for the phospho-p38 MAPK and total p38 MAPK, phospho-Akt and total Akt, phospho-ERK and total ERK. They were visualized with appropriate HRP-conjugated secondary Abs (Southern Biotechnology Associates, Birmingham, AL) and an ECL detection system (Pierce, Rockford, IL). When inhibitors were used, cells were incubated for 30 min before addition of trimeric CD40L.

Statistical analysis

Data were compared using the Student t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CD40L activates p38 MAPK, ERK, and Akt in immature MDDCs

Signal transduction via the MAPK such as ERKs and the p38 MAPK or Akt kinase, a downstream target of the PI3K, play important roles in cellular responses including cell proliferation, differentiation, and survival. CD40 ligation of immature MMDCs results in a potent maturational and survival stimulus. We therefore looked for activation of these signaling pathways in human immature MDDCs treated with trimeric CD40L (CD154). We elected to study MDDCs obtained from individuals with detectable viral-specific CD8+ T cell responses so that we could also assess the functional activity of these DC on autologous CTL responses (see below). These included healthy human volunteers with CTL directed against EBV epitopes (participants 1–3) and HIV-1-infected individuals with CTL responses directed against HIV-1 and EBV epitopes (participants 4–6, as described in Materials and Methods). In addition, we have previously shown that immature MDDC obtained from HIV-1-infected and HIV-1-uninfected individuals undergo maturation to a similar degree when treated with trimeric CD40L (14). Monocytes isolated from PBMC were cultured with complete RPMI 1640 medium in the presence of GM-CSF and IL-4 for 7 days to generate immature MDDCs, which were then exposed to trimeric CD40L. Activation of PI3K, ERK, or p38 MAPK results in their phosphorylation. We found that within 30 min of CD40L conditioning of immature MDDCs, PI3K, ERK, and p38 MAPK were activated (Fig. 1). It has previously been shown that, the PI3K can be specifically inhibited by the chomone LY294002 (49), the ERK pathway can be specifically inhibited by the flavonoid, PD98059 (50), and the p38 MAPK can be specifically inhibited by the imidazole SB203580 (51). Pretreatment of MDDCs with the PI3K inhibitor LY294002, ERK inhibitor PD98059, or p38 MAPK inhibitor SB203580 blocked phosphorylation of PI3K, ERK, or p38 MAPK, respectively, in response to CD40L stimulation (Fig. 1). Thus, the PI3K, ERK, and p38 MAPK signaling pathways are activated after CD40 signaling of human immature MDDCs.



View larger version (67K):
[in this window]
[in a new window]
  		FIGURE 1. CD40L induces the phosphorylation of p38 MAPK, ERK, and Akt. Immature MDDCs from participant 1 were exposed to trimeric CD40L (2 µg/ml) for indicated time intervals at 37°C and then subjected to Western blot analysis with specific Abs. In some cases, the immature MDDCs were preincubated with signal tranduction pathway inhibitors for 30 min before exposure to CD40L. The left side shows the detection of phosphorylated forms of p38 MAPK, AKT, and ERK, and the right side shows the same plots probed for total p38 MAPK, AKT, and ERK proteins, respectively, to demonstrate equal loading of samples. Phosphorylation of p38 MAPK, AKT, and ERK induced within 30 min and persisted for 60 min after exposure of immature MDDCs to CD40L. The phosphorylation of p38 MAPK, AKT, or ERK are inhibited if immature MDDCs are preincubated with their corresponding inhibitor SB203580, LY294002, or PD98059, respectively. SB203580, LY294002, and PD98059 were all dissolved in DMSO; therefore, immature MDDCs that had been incubated with DMSO alone were used as controls. Similar results were obtained in three separate experiments. d, DMSO control.

 
Roles of p38 MAPK, ERK, and PI3K pathways in MDDCs maturation and activation

Maturation of MDDC with CD40L leads to up-regulation of cell surface CD80, CD83, CD86, and HLA-DR, and down-regulation of CD1a. We examined whether p38 MAPK, ERK, and PI3K pathways are necessary for CD40L-induced surface phenotype changes on MDDCs. Inhibition of ERK activation with inhibitor PD98059 slightly reduced HLA-DR and CD86, which was not statistically significant, and had no effect on any other surface phenotype measures of MDDC maturation in response to CD40L stimulation (Fig. 2). Blocking the p38 MAPK pathway with the inhibitor SB203580, however, significantly inhibited the CD40L-induced up-regulation of CD83 and CD86 (p < 0.05). Inhibition of p38 MAPK slightly reduced CD40L induction of HLA-DR and CD80 (not significant), and did not affect the CD40L-induced down-regulation of CD1a (Fig. 2). Blocking of the PI3K pathway minimally inhibited CD40L-induced CD1a down-regulation and minimally inhibited CD40L-induced up-regulation of CD80, CD83, CD86, and HLA-DR but this was not statistically significant (Fig. 2). These results show that only certain features of MDDC maturation, such as CD83 and CD86 expression, are regulated by p38 MAPK. Because inhibition of p38 MAPK did not entirely abrogate CD83/CD86 expression after CD40L treatment, as observed with the DMSO alone condition, this suggests that other signaling pathways also play a role in CD83/CD86 up-regulation. Although, inhibiting ERK and PI3K slightly alters CD40L-induced surface marker changes, they do not appear to play a significant role in their regulation.



View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 2. Roles of p38 MAPK, ERK, and PI3K pathways in MDDCs maturation and activation. A representative experiment from HIV-1-infected participant 3 (A) is shown. Immature MDDCs were incubated at 37°C for 72 h with medium plus DMSO (negative control), or trimeric CD40L (2 µg/ml). When the inhibitors were used, MDDCs were preincubated with indicated inhibitors for 30 min before addition of CD40L. MDDCs were harvested and expression of surface molecules was assessed by flow cytometric analysis using mouse anti-human mAbs: PE-conjugated CD80 and CD83, FITC-conjugated CD1a and CD86, and allophycocyanin-conjugated HLA-DR. Appropriate fluorochrome-conjugated isotype was used as control for each stain. Values represent the mean fluorescence intensity subtracted from the value of matched isotype control mouse mAbs (dotted histogram). Data are analyzed using FlowJo software. B, Summary data from experiments performed on MDDCs obtained from all four participants are shown. Bars represent SE on the mean. Comparisons were performed between all conditions and CD40L+DMSO. Asterisk indicates statistically significant (p < 0.05) associations between signaling inhibitor and CD40L conditions. In all experiments, CD40L+DMSO condition was always statistically significant (p < 0.05) from DMSO alone conditions (unconditioned MDDC). MFI, mean fluorescent intensity.

 
Roles of p38 MAPK, ERK, and PI3K pathways in DC-induced allogeneic T cell responses

Activated DCs are potent APCs that sensitize T lymphocytes to alloantigens and normal Ags. We therefore tested the ability of CD40L-activated MDDCs to stimulate T lymphocytes in allogeneic MLRs. As reported previously, CD40L-stimulated MDDCs strongly enhanced allogeneic T cell proliferation when compared with immature MDDCs (52). Blocking the ERK pathway with PD98059 or PI3K pathway with LY294002 slightly reduced the capacity of CD40L-induced allogeneic T cell proliferation (Fig. 3). Inhibition of p38 MAPK pathway with SB203580, however, strongly reduced the capacity of CD40L-induced allogeneic T cell proliferation (Fig. 3) to the level of allogeneic responses with unconditioned immature MDDCs (Fig. 3, p < 0.05). Thus, the p38 MAPK pathway plays an important role in the allostimulatory capacity of CD40L-activated MDDCs.



View larger version (27K):
[in this window]
[in a new window]
  		FIGURE 3. Effects of signaling transduction via p38 MAPK, ERK, or PI3K pathways on allostimulatory ability of CD40L-activated MDDCs. Immature MDDCs, not pretreated or pretreated with indicated inhibitors for 30 min before addition of CD40L, were stimulated with CD40L (2 µg/ml) for 3 days, then plated into 96-well round-bottom tissue-culture plates with allogeneic T lymphocytes at a ratio of 1:20 (MDDCs to T cells). Cells were cocultured for 72 h and pulsed with 2 µCi (0.076 MBq) [3H]thymidine for an additional 6 h. [3H]Thymidine incorporation in harvested cells was assessed by liquid scintillation spectrometry. These data are mean ± SEM of triplicates and representative of four independent experiments with different donors. CD40L, trimeric CD40; LY, LY294002; SB, SB203580; PD, PD98059.

 
Roles of p38 MAPK, ERK, and PI3K pathways in the expansion of viral-specific memory CTL responses induced by CD40L-activated MDDCs

To determine the role of the signal transduction via p38 MAPK, ERK, or PI3K in the expansion of virus specific memory CD8+ CTL responses induced by CD40L-activated MDDCs, we used an in vitro coculture method in which peptide pulsed conditioned MDDCs are cocultured with CD8+ T cells for 7 days in the absence of exogenous cytokines as previously described (14). Three healthy HIV-1-uninfected (participants 1–3) and three asymptomatic HIV-1-infected (participants 4–6) individuals who previously demonstrated EBV- or HIV-1-specific memory responses to well-described HLA-restricted CTL epitopes were studied (also described in Materials and Methods). Circulating virus-specific IFN-{gamma}-producing memory cells within PBMCs, as determined by ELISPOT assay, ranged from 250 to 1000 spot forming cells/106 PBMC in these six study individuals (data not shown). Immature MDDCs derived from these individuals were conditioned with trimeric CD40L for 3 days, then pulsed with the HLA-restricted peptides that they responded to previously (see Materials and Methods) and cocultured with autologous PBMCs for 7 days. For signaling inhibitor experiments, MDDCs were preincubated with indicated inhibitors for 30 min, and then incubated with 2 µg/ml trimeric CD40L. DMSO was used as a control because the inhibitors were dissolved in DMSO. After 7 days of coculture, CTL effector activity was assessed by measuring intracellular IFN-{gamma} production after exposure to peptide-pulsed targets (autologous B lymphoblastoid cell lines or autologous T cell-depleted PBMCs). It should be noted that only MDDCs were exposed to inhibitors before conditioning and that effector CD8+ T cells from PBMCs were added later, in the absence of any inhibitors. A representative experiment measuring CTL by IFN-{gamma} by intracellular flow cytometry from HIV-1 seropositive participant 6 is illustrated in Fig. 4A. A summary of the IFN-{gamma} flow cytometric data from participants 1–5 are illustrated in Fig. 4B. Summary pooled data from all participants are shown in Fig. 4C. Immature MDDCs treated with CD40L significantly enhanced EBV- or HIV-1-specific memory CTL responses when compared with medium-treated immature MDDCs (p < 0.0009) (Fig. 4). Blocking the PI3K pathway with LY294002 or ERK pathway with PD98059 slightly reduced the expansion of EBV- or HIV-1-specific viral memory CTLs in response to CD40L-activated MDDCs by ~10–20%, which was statistically significant (p < 0.03 and p < 0.04, respectively; Fig. 4C). Inhibition of the p38 MAPK pathway with SB203580, however, markedly inhibited the capacity of CD40L-activated DCs in the expansion of EBV- or HIV-1-specific viral CTL responses (p < 0.007; Fig. 4C). The effects of SB203580 were not due to nonspecific cellular toxicity as viability of PBMCs treated with SB203580 was comparable to untreated cells by trypan blue staining (data not shown). Inhibition of p38 MAPK within MDDCs almost completely reversed the effects of trimeric CD40L to expand CTL as the CTL response reached to the level of that using unconditioned immature MDDCs (Fig. 4C). Thus, p38 MAPK pathway plays a critical role in the expansion of EBV- or HIV-1-specific viral CTL responses induced by CD40L-activated MDDCs.



View larger version (25K):
[in this window]
[in a new window]
  		FIGURE 4. Effect of p38 MAPK, ERK, and PI3K pathway inhibitors in the expansion of viral-specific memory CTL responses induced by CD40L-activated MDDCs. A, PBMCs from an HIV-1-infected individual (participant 6) were cocultured with MDDCs that were either nonpulsed (far left) or pulsed with HLA-restricted peptides of HIV-1 proteins. In certain conditions, MDDCs were previously treated with CD40L (2 µg/ml) for 72 h in the presence or absence of indicated inhibitors. On day 7, HIV-1-specific CTL activity was assessed by intracellular flow cytometric analysis of IFN-{gamma}-producing CD8+ T cells. The experiment with participant 6 was repeated with similar results. In all conditions with CD40L, DCs were always pulsed with peptide. B, Summary intracellular flow cytometric analysis of EBV-specific CTL from three healthy individuals (participants 1, 2, and 3) and one HIV-1-infected individual (participant 5) and HIV-1-specific CTL from an HIV-1-infected individual (participant 4) are graphically depicted. C, Pooled data from all participants are shown comparing all conditions (except DC without peptide) to the CD40L alone condition, as this latter condition gave maximal CTL. *, p < 0.05. Statistical comparisons among data pooled from all participants were performed using the following culture conditions: DC vs DCp, p < 0.0006 (data not shown); DCp vs CD40L, p < 0.0009; CD40L vs CD40L+LY, p < 0.03; CD40L vs CD40L+PD, p < 0.04; CD40L vs CD40L+SB, p < 0.007; CD40L+SB vs DCp, p = not significant. DC, nonpeptide-pulsed DC; DCp, DC pulsed with peptide; CD40L, trimeric CD40L; LY, PI3K inhibitor LY294002; PD, ERK inhibitor PD98059; SB, p38 MAPK inhibitor SB203580. All inhibitors were used in a final concentration of 25 µM.

 
Inhibition of p38 MAPK, ERK, or PI3K leads to decreased survival of CD40L-activated MDDCs

Consistent with previous reports, immature MDDCs undergo spontaneous apoptosis in a 48-h culture (39, 40, 53), which can be inhibited by the addition of trimeric CD40L (11). Accordingly, we addressed whether the activation of p38 MAPK, PI3K, or ERK is involved in CD40L-mediated MDDC survival. As shown in Fig. 5, A and B, trimeric CD40L significantly inhibited early MDDC spontaneous apoptosis, as defined by annexin V or activated caspase 3 expression. Inhibition of either p38 MAPK, PI3K, or ERK significantly reversed the CD40L-induced MDDCs survival (pooled data, all p < 0.05, also Fig. 5D), albeit to differing degrees. Whereas p38 MAPK and ERK inhibitors had moderate effects on cellular survival, the PI3K inhibitor completely blocked the CD40L-induced inhibition of apoptosis, indicating that PI3K/Akt is a major signaling pathway involved in CD40L-induced cellular survival (Fig. 5). These effects were seen for both annexin V and activated caspase 3 expression. In fact, blocking PI3K signaling pathway not only completely reversed CD40L-induced inhibition of apoptosis, but tended to produce more apoptosis than unconditioned immature MDDCs (Fig. 5). To ensure that the effects of PI3K inhibitor LY294002 were not from nonspecific cellular cytotoxicity, we found that PBMC treated with or without LY294002 showed no statistical difference in cell apoptosis identified by annexin V (data not shown).



View larger version (31K):
[in this window]
[in a new window]
  		FIGURE 5. Inhibition of ERK, PI3K, or p38 MAPK leads to decreased survival of trimeric CD40L-conditioned MDDCs. A representative experiment from participant 6 is shown. Immature MDDCs, not pretreated or pretreated with indicated inhibitors for 30 min, were incubated for 48 h with 2 µg/ml trimeric CD40L or medium alone. Cells from each condition were harvested and aliquoted for flow cytometric analysis for Annexin VFITC or HLA-DR-allophycocyanin/caspase-3FITC/Bcl-2PE staining. A, Apoptotic cells were assessed for surface phosphatidylserine on the outer leaflet of apoptotic cell membranes using annexin V-fluorescein staining. The numbers (inset right) represents percentage of Annexin VFITC-positive apoptotic MDDCs per total MDDCs. B, Caspase 3 activation of DC was determined by gating on HLA-DR-positive cells (as DC strongly express HLA-DR). The number in each box represents percentage of positive caspase 3 MDDCs per total HLA-DR-positive cells. C, Intracellular Bcl-2 expression is represented. The number (inset in box) in each condition represents percentage of positive Bcl-2 cells per total HLA-DR-positive cells. A representative experiment of four experiments for annexin V, caspase 3, and Bcl-2 analysis is shown. D, Pooled data from four participants studied is shown for annexin V staining. *, p < 0.05. Statistical analyses of pooled data were performed on the following conditions: DMSO vs DMSO+CD40L, p < 0.05; DMSO+CD40L vs CD40L+LY294002, p < 0.004; DMSO+CD40L vs CD40L + PD098059, p < 0.009; DMSO+CD40L vs CD40L + SB203580, p < 0.003.

 
To understand the potential mechanisms by which CD40L-promoted MDDC survival, we examined Bcl-2 expression, a negative regulator of apoptosis. We found that treatment with CD40L significantly enhanced expression of Bcl-2 in MDDCs (Fig. 5C). Bcl-2 up-regulation by CD40L stimulation could be significantly blocked by p38 MAPK, ERK, or PI3K inhibitor, but to the greatest degree by the PI3K inhibitor (Fig. 5C, pooled data, all p < 0.05).

In summary, CD40L-induced suppression of apoptosis of MDDCs is mediated predominantly by the PI3K/Akt pathway, but can also to a lesser degree be mediated by the ERK and p38 MAPK pathways. CD40L-induced suppression of MDDC apoptosis can, in part, be explained by inhibition of caspase 3 activation and an enhancement of Bcl-2 expression, all of which are regulated by the PI3K/Akt, ERK, and p38 MAPK pathways.

Effects of p38 MAPK, ERK, or PI3K inhibitors on CD40L-induced IL-12 production from MDDCs

IL-12 is a pleiotropic cytokine that is secreted by activated professional APCs. IL-12 can induce Th1-type cellular responses, T cell proliferation, and IFN-{gamma} secretion from activated T cells and NK cells. In addition, it has been reported that IL-12 prevents apoptotic cell death of the activated human and mouse peripheral T cells (12, 17, 38, 54, 55). We and others previously have demonstrated that MDDCs could be induced to produce IL-12 after CD40 ligation (11, 12, 14). We thus evaluated the relative roles of PI3K, p38 MAPK, and ERK in IL-12 production of CD40L-stimulated MDDCs. Immature MDDCs were stimulated for 16 h with CD40L, and IL-12 induction was measured by intracellular flow cytometric analysis. A representative experiment is illustrated in Fig. 6A and summary data from all experiments is shown in Fig. 6B. CD40L stimulation induced significant amounts of IL-12 production by MDDCs. Blocking the ERK signaling pathway with PD98059 had no effects on the IL-12 production whereas blocking either PI3K with LY294002 or p38 MAPK with SB203580 significantly reduced the effect of CD40L on IL-12 production by ~50%. Blocking the p38 MAPK signaling pathway, however, gave the strongest inhibition of IL-12 production. Thus, both the p38 MAPK and PI3K pathways play a critical role in the induction of IL-12 through CD40 ligation of MDDC.



View larger version (36K):
[in this window]
[in a new window]
  		FIGURE 6. Effects of ERK, PI3K, or p38 MAPK inhibitor on CD40L-induced IL-12 production of MDDCs. Immature MDDCs, not pretreated or pretreated with indicated inhibitors for 30 min before addition of CD40L, and stimulated with CD40L (2 µg/ml) at 37°C for 16 h. Monensin was added during the last 6 h incubation. IL-12 production was determined by intracellular staining using mouse anti-human IL-12 (p40/p70) Ab. The conditioned MDDCs were also counterstained for HLA-DR. The numbers (inset upper right) represent the percentage of IL-12 p40/p70-positive MDDCs per total MDDCs. Data are taken from HIV-1-infected participant 3 and are representative of experiments with MDDCs derived from HIV-1-infected and HIV-1-uninfected individuals. Experiments were performed with or without priming with 100 ng/ml IFN-{gamma} and similar results were observed. B, Pooled data from all participants are shown. *, p < 0.05, with the following statistical comparisons performed: DMSO vs DMSO+CD40L, p < 0.005; DMSO+CD40L vs CD40L + LY294002, p < 0.01; DMSO+CD40L vs CD40L + PD098059, p = 0.17 (not significant); DMSO+CD40L vs CD40L + SB203580, p < 0.004.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
During the development of an ongoing immune response, activated CD4+ T cells express CD40 ligand which in turn stimulate immature DCs through CD40 to become superactivated and optimally prime CD8+ T cells as well as help B cells. Our data show that at least three signaling pathways are activated within immature MDDCs after CD40 ligation; the PI3K, ERK, and p38 MAPK pathways. It was surprising that surface markers of activation and maturation were not greatly affected by the inhibition of these pathways. Only the p38 MAPK pathway played a significant role in CD83 and CD86 up-regulation, although inhibiting p38 MAPK did not completely abrogate CD83 and CD86 expression. This indicates that other signaling pathways, likely are involved with regulation of CD1a, CD80, and HLA-DR, as well as CD83 and CD86. The role of CD83 in inducing immune responses is beginning to emerge. Recently, Ledbetter and colleagues (56) showed that CD83, similar to CD80 and CD86, likely plays a costimulatory role in the induction of MLRs and proliferation of specific CD8+ T cell responses. Our findings are similar to those of Ardeshna et al. (40) who showed that inhibiting p38 MAPK prevented LPS-induced up-regulation of CD80, CD83, and CD86, but had no effect on other surface markers of LPS-induced DC activation and maturation. These studies also showed that NF-{kappa}B played an important role in the up-regulation of HLA-DR, CD80, CD83, and CD86 in LPS-treated DCs. We, however, did not examine the NF-{kappa}B pathway in this study.

Previous work has shown that p38 MAPK plays a major role in DC IL-12 expression after CD40 receptor ligation (34). We find that both p38 MAPK and PI3K play a role in CD40L-mediated IL-12 induction by DCs, with p38 MAPK pathway making the largest contribution. Interestingly, in a murine model, Fukao et al. (57) demonstrated that the PI3K pathway negatively regulated IL-12 production. This indicates that comparing signaling pathways between murine and human DC systems may not always be comparable, as previously demonstrated (58).

All of p38 MAPK, ERK, and PI3K pathways played a role in CD40L-mediated MDDC survival, with PI3K being the most important. In fact, inhibiting the PI3K pathway in CD40L-stimulated MDDCs resulted in apoptosis exceeding that of unconditioned MDDCs. We showed that these pathways mediate CD40L-induced survival, albeit to varying degrees, by inhibiting caspase 3 activation and increasing Bcl-2 production. The PI3K pathway, has been most extensively studied, and has been shown to inhibit apoptosis by phosphorylating Akt downstream, which phosphorylates and inactivates the proapoptotic factor BAD, caspase 9 and thus subsequent caspase 3 activation, and the Forkhead family of transcription factors, which are involved in the expression of death genes (41). Although p38 MAPK and ERK pathways have been shown to induce apoptosis in T cells (36), these pathways have been shown to inhibit apoptosis in LPS-treated murine DCs; p38 MAPK by inducing the cellular inhibitor of apoptosis protein 2, cIAP2, and ERK by an undefined mechanism (34, 39). Also, the mechanisms as to how these signaling pathways enhance Bcl-2 production are currently unknown.

We also assessed the role of p38 MAPK, ERK, and PI3K pathways on the functional activity of CD40L-treated MDDC by examining allogeneic and virus-specific T cell responses. Although each of these pathways influence various features of CD40L-induced MDDC maturation, only inhibition of p38 MAPK could completely reverse the ability of CD40L-conditioned MDDC to stimulate allogeneic or virus-specific CD8+ T cell responses. This is not surprising, given that the p38 MAPK pathway is involved in many aspects of the innate and adaptive immune response (36). We were unable to identify a unique downstream event of p38 MAPK signaling in MDDCs that was instrumental for DC functional capacity. Our observations indicate that p38 MAPK is required for a number of features involved in DC maturation including up-regulation of CD83 and CD86, IL-12 production, and inhibition of apoptosis. It is probable that this combination of events is necessary to optimally prime T cells, rather than one feature alone. It was somewhat surprising to find that, although the PI3K pathway was most critical in the prevention of MDDC apoptosis, inhibition of this pathway could still activate T cells to varying degrees. Because our MDDC-T cell coculture system only stimulated cells for a 7-day period, it is possible that the effects of PI3K inhibition might have been more dramatic on T cell expansion with longer culture periods or in in vivo systems, in which the longevity of DCs becomes more important in maintaining immune responses.

In summary, the p38 MAPK pathway is necessary for CD40L to condition DCs to become competent Ag presenters to T cells. Recently, a number of small molecule inhibitors of the p38 MAPK pathway have been proposed for use in the clinical setting to prevent inflammatory responses (59, 60). It is likely that these inhibitors will partly work by preventing the necessary signaling pathways provided by CD40L to optimally mature and activate DCs.


   Acknowledgments
 
We thank our patients for their time and commitment. We thank Jason Ho and Susan Moir for helpful comments and suggestions.


   Footnotes
 
1 This work was supported through a grant by Aventis Pasteur-University of Toronto Research Program. Q.Y. is a career scientist of the Ontario HIV Treatment Network, and M.O. is a career scientist of the Canadian Institutes of Health Research. Back

2 Address correspondence and reprint requests to Dr. Mario A. Ostrowski, Clinical Sciences Division, Medical Sciences Building, Room 6271, 1 King’s College Circle, University of Toronto, Toronto, Ontario M5S 1A8, Canada. E-mail address: m.ostrowski{at}utoronto.ca Back

3 Abbreviations used in this paper: DC, dendritic cell; CD40L, CD40 ligand; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; PI3K, phosphoinositide-3-OH kinase; MDDC, monocyte-derived DC; TRAF, TNFR-associated factor; JNK, c-jun N-terminal kinase. Back

Received for publication September 23, 2003. Accepted for publication March 4, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Reusser, P., G. Cathomas, R. Attenhofer, M. Tamm, G. Thiel, M. J. Reddehase, U. H. Koszinowski. 1999. Cytomegalovirus (CMV)-specific T cell immunity after renal transplantation mediates protection from CMV disease by limiting the systemic virus load: significance of herpesvirus immediate early gene expression in cellular immunity to cytomegalovirus infection. J. Infect. Dis. 180:247.[Medline]
  2. Callan, M. F., L. Tan, N. Annels, G. S. Ogg, J. D. Wilson, C. A. O’Callaghan, N. Steven, A. J. McMichael, A. B. Rickinson. 1998. Direct visualization of antigen-specific CD8+ T cells during the primary immune response to Epstein-Barr virus in vivo. J. Exp. Med. 187:1395.[Abstract/Free Full Text]
  3. Pantaleo, G., J. F. Demarest, T. Schacker, M. Vaccarezza, O. J. Cohen, M. Daucher, C. Graziosi, S. S. Schnittman, T. C. Quinn, G. M. Shaw, et al 1997. The qualitative nature of the primary immune response to HIV infection is a prognosticator of disease progression independent of the initial level of plasma viremia. Proc. Natl. Acad. Sci. USA 94:254.[Abstract/Free Full Text]
  4. Banchereau, J., R. M. Steinman. 1998. Dendritic cells and the control of immunity. Nature 392:245.[Medline]
  5. Cella, M., M. Salio, Y. Sakakibara, H. Langen, I. Julkunen, A. Lanzavecchia. 1999. Maturation, activation, and protection of dendritic cells induced by double-stranded RNA. J. Exp. Med. 189:821.[Abstract/Free Full Text]
  6. Caux, C., C. Massacrier, B. Vanbervliet, B. Dubois, C. Van Kooten, I. Durand, J. Banchereau. 1994. Activation of human dendritic cells through CD40 cross-linking. J. Exp. Med. 180:1263.[Abstract/Free Full Text]
  7. Hacker, H., H. Mischak, T. Miethke, S. Liptay, R. Schmid, T. Sparwasser, K. Heeg, G. B. Lipford, H. Wagner. 1998. CpG-DNA-specific activation of antigen-presenting cells requires stress kinase activity and is preceded by non-specific endocytosis and endosomal maturation. EMBO J. 17:6230.[Medline]
  8. De Smedt, T., B. Pajak, E. Muraille, J. Urbain, O. Leo, M. Moser. 1997. Positive and negative regulation of dendritic cell function by lipopolysaccharide in vivo. Adv. Exp. Med. Biol. 417:535.[Medline]
  9. Sallusto, F., A. Lanzavecchia. 1994. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin-4 and downregulated by tumor necrosis factor-{alpha}. J. Exp. Med. 179:1109.[Abstract/Free Full Text]
  10. Ignatius, R., M. Marovich, E. Mehlhop, L. Villamide, K. Mahnke, W. I. Cox, F. Isdell, S. S. Frankel, J. R. Mascola, R. M. Steinman, M. Pope. 2000. Canarypox virus-induced maturation of dendritic cells is mediated by apoptotic cell death and tumor necrosis factor {alpha} secretion. J. Virol. 74:11329.[Abstract/Free Full Text]
  11. Yu, Q., J. X. Gu, C. Kovacs, J. Freedman, E. K. Thomas, M. A. Ostrowski. 2003. Cooperation of TNF family members CD40 ligand, RANKL, and TNF{alpha} in the activation of dendritic cells and the expansion of viral specific CD8+ T cell memory responses in HIV-1-infected and -uninfected individuals. J. Immunol. 170:1797.[Abstract/Free Full Text]
  12. Cella, M., D. Scheidegger, K. Palmer-Lehmann, P. Lane, A. Lanzavecchia, G. Alber. 1996. Ligation of CD40 on dendritic cells triggers production of high levels of interleukin-12 and enhances T cell stimulatory capacity: T-T help via APC activation. J. Exp. Med. 184:747.[Abstract/Free Full Text]
  13. Lapointe, R., J. F. Toso, C. Butts, H. A. Young, P. Hwu. 2000. Human dendritic cells require multiple activation signals for the efficient generation of tumor antigen-specific T lymphocytes. Eur. J. Immunol. 30:3291.[Medline]
  14. Ostrowski, M. A., S. J. Justement, L. Ehler, S. B. Mizell, S. Lui, J. Mican, B. D. Walker, E. K. Thomas, R. Seder, A. S. Fauci. 2000. The role of CD4+ T cell help and CD40 ligand in the in vitro expansion of HIV-1-specific memory cytotoxic CD8+ T cell responses. J. Immunol. 165:6133.[Abstract/Free Full Text]
  15. Guo, Y., Y. Wu, S. Shinde, M. S. Sy, A. Aruffo, Y. Liu. 1996. Identification of a costimulatory molecule rapidly induced by CD40L as CD44H. J. Exp. Med. 184:955.[Abstract/Free Full Text]
  16. Kennedy, M. K., K. S. Picha, W. C. Fanslow, K. H. Grabstein, M. R. Alderson, K. N. Clifford, W. A. Chin, K. M. Mohler. 1996. CD40/CD40 ligand interactions are required for T cell-dependent production of interleukin-12 by mouse macrophages. Eur. J. Immunol. 26:370.[Medline]
  17. Koch, F., U. Stanzl, P. Jennewein, K. Janke, C. Heufler, E. Kampgen, N. Romani, G. Schuler. 1996. High level IL-12 production by murine dendritic cells: upregulation via MHC class II and CD40 molecules and downregulation by IL-4 and IL-10. J. Exp. Med. 184:741.[Abstract/Free Full Text]
  18. Kuniyoshi, J. S., C. J. Kuniyoshi, A. M. Lim, F. Y. Wang, E. R. Bade, R. Lau, E. K. Thomas, J. S. Weber. 1999. Dendritic cell secretion of IL-15 is induced by recombinant huCD40LT and augments the stimulation of antigen-specific cytolytic T cells. Cell. Immunol. 193:48.[Medline]
  19. Mackey, M. F., J. R. Gunn, C. Maliszewsky, H. Kikutani, R. J. Noelle, R. J. Barth, Jr. 1998. Dendritic cells require maturation via CD40 to generate protective antitumor immunity. J. Immunol. 161:2094.[Abstract/Free Full Text]
  20. Yang, Y., J. M. Wilson. 1996. CD40 ligand-dependent T cell activation: requirement of B7-CD28 signaling through CD40. Science 273:1862.[Abstract/Free Full Text]
  21. Schoenberger, S. P., R. E. Toes, E. I. van der Voort, R. Offringa, C. J. Melief. 1998. T-cell help for cytotoxic T lymphocytes is mediated by CD40-CD40L interactions. Nature 393:480.[Medline]
  22. Ridge, J. P., F. Di Rosa, P. Matzinger. 1998. A conditioned dendritic cell can be a temporal bridge between a CD4+ T-helper and a T-killer cell. Nature 393:474.[Medline]
  23. Hermans, I. F., D. S. Ritchie, A. Daish, J. Yang, M. R. Kehry, F. Ronchese. 1999. Impaired ability of MHC class II dendritic cells to provide tumor protection is rescued by CD40 ligation. J. Immunol. 163:77.[Abstract/Free Full Text]
  24. Bennett, S. R., F. R. Carbone, F. Karamalis, R. A. Flavell, J. F. Miller, W. R. Heath. 1998. Help for cytotoxic-T-cell responses is mediated by CD40 signalling. Nature 393:478.[Medline]
  25. Oxenius, A., K. A. Campbell, C. R. Maliszewski, T. Kishimoto, H. Kikutani, H. Hengartner, R. M. Zinkernagel, M. F. Bachmann. 1996. CD40-CD40 ligand interactions are critical in T-B cooperation but not for other anti-viral CD4+ T cell functions. J. Exp. Med. 183:2209.[Abstract/Free Full Text]
  26. Bachmann, M. F., B. R. Wong, R. Josien, R. M. Steinman, A. Oxenius, Y. Choi. 1999. TRANCE, a tumor necrosis factor family member critical for CD40 ligand-independent T helper cell activation. J. Exp. Med. 189:1025.[Abstract/Free Full Text]
  27. Whitmire, J. K., M. K. Slifka, I. S. Grewal, R. A. Flavell, R. Ahmed. 1996. CD40 ligand-deficient mice generate a normal primary cytotoxic Tlymphocyte response but a defective humoral response to a viral infection. J. Virol. 70:8375.[Abstract]
  28. Lu, Z., L. Yuan, X. Zhou, E. Sotomayor, H. I. Levitsky, D. M. Pardoll. 2000. CD40-independent pathways of T cell help for priming of CD8+ cytotoxic T lymphocytes. J. Exp. Med. 191:541.[Abstract/Free Full Text]
  29. Grammer, A. C., P. E. Lipsky. 2000. CD40-mediated regulation of immune responses by TRAF-dependent and TRAF-independent signaling mechanisms. Adv. Immunol. 76:61.[Medline]
  30. Kuhne, M. R., M. Robbins, J. E. Hambor, M. F. Mackey, Y. Kosaka, T. Nishimura, J. P. Gigley, R. J. Noelle, D. M. Calderhead. 1997. Assembly and regulation of the CD40 receptor complex in human B cells. J. Exp. Med. 186:337.[Abstract/Free Full Text]
  31. Bishop, G. A., B. S. Hostager. 2003. The CD40-CD154 interaction in B cell-T cell liaisons. Cytokine Growth Factor Rev. 14:297.[Medline]
  32. Vidalain, P. O., O. Azocar, C. Servet-Delprat, C. Rabourdin-Combe, D. Gerlier, S. Manie. 2000. CD40 signaling in human dendritic cells is initiated within membrane rafts. EMBO J. 19:3304.[Medline]
  33. Garceau, N., Y. Kosaka, S. Masters, J. Hambor, R. Shinkura, T. Honjo, R. J. Noelle. 2000. Lineage-restricted function of nuclear factor {kappa}B-inducing kinase (NIK) in transducing signals via CD40. J. Exp. Med. 191:381.[Abstract/Free Full Text]
  34. Aicher, A., G. L. Shu, D. Magaletti, T. Mulvania, A. Pezzutto, A. Craxton, E. A. Clark. 1999. Differential role for p38 mitogen-activated protein kinase in regulating CD40-induced gene expression in dendritic cells and B cells. J. Immunol. 163:5786.[Abstract/Free Full Text]
  35. Arron, J. R., M. Vologodskaia, B. R. Wong, M. Naramura, N. Kim, H. Gu, Y. Choi. 2001. A positive regulatory role for Cbl family proteins in tumor necrosis factor-related activation-induced cytokine (TRANCE) and CD40L-mediated Akt activation. J. Biol. Chem. 276:30011.[Abstract/Free Full Text]
  36. Dong, C., R. J. Davis, R. A. Flavell. 2002. MAP kinases in the immune response. Annu. Rev. Immunol. 20:55.[Medline]
  37. Mackey, M. F., Z. Wang, K. Eichelberg, R. N. Germain. 2003. Distinct contributions of different CD40 TRAF binding sites to CD154-induced dendritic cell maturation and IL-12 secretion. Eur. J. Immunol. 33:779.[Medline]
  38. Macatonia, S. E., N. A. Hosken, M. Litton, P. Vieira, C. S. Hsieh, J. A. Culpepper, M. Wysocka, G. Trinchieri, K. M. Murphy, A. O’Garra. 1995. Dendritic cells produce IL-12 and direct the development of Th1 cells from naive CD4+ T cells. J. Immunol. 154:5071.[Abstract]
  39. Rescigno, M., M. Martino, C. L. Sutherland, M. R. Gold, P. Ricciardi-Castagnoli. 1998. Dendritic cell survival and maturation are regulated by different signaling pathways. J. Exp. Med. 188:2175.[Abstract/Free Full Text]
  40. Ardeshna, K. M., A. R. Pizzey, S. Devereux, A. Khwaja. 2000. The PI3 kinase, p38 SAP kinase, and NF-{kappa}B signal transduction pathways are involved in the survival and maturation of lipopolysaccharide-stimulated human monocyte-derived dendritic cells. Blood 96:1039.[Abstract/Free Full Text]
  41. Brunet, A., S. R. Datta, M. E. Greenberg. 2001. Transcription-dependent and -independent control of neuronal survival by the PI3K-Akt signaling pathway. Curr. Opin. Neurobiol. 11:297.[Medline]
  42. Goyal, A., Y. Wang, M. M. Graham, A. I. Doseff, N. Y. Bhatt, C. B. Marsh. 2002. Monocyte survival factors induce Akt activation and suppress caspase-3. Am. J. Respir. Cell Mol. Biol. 26:224.[Abstract/Free Full Text]
  43. Brunet, A., A. Bonni, M. J. Zigmond, M. Z. Lin, P. Juo, L. S. Hu, M. J. Anderson, K. C. Arden, J. Blenis, M. E. Greenberg. 1999. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 96:857.[Medline]
  44. Kelley, T. W., M. M. Graham, A. I. Doseff, R. W. Pomerantz, S. M. Lau, M. C. Ostrowski, T. F. Franke, C. B. Marsh. 1999. Macrophage colony-stimulating factor promotes cell survival through Akt/protein kinase B. J. Biol. Chem. 274:26393.[Abstract/Free Full Text]
  45. Kennedy, S. G., A. J. Wagner, S. D. Conzen, J. Jordan, A. Bellacosa, P. N. Tsichlis, N. Hay. 1997. The PI 3-kinase/Akt signaling pathway delivers an anti-apoptotic signal. Genes Dev. 11:701.[Abstract/Free Full Text]
  46. Borrow, P., A. Tishon, S. Lee, J. Xu, I. S. Grewal, M. B. Oldstone, R. A. Flavell. 1996. CD40L-deficient mice show deficits in antiviral immunity and have an impaired memory CD8+ CTL response. J. Exp. Med. 183:2129.[Abstract/Free Full Text]
  47. Borrow, P., D. F. Tough, D. Eto, A. Tishon, I. S. Grewal, J. Sprent, R. A. Flavell, M. B. Oldstone. 1998. CD40 ligand-mediated interactions are involved in the generation of memory CD8+ cytotoxic T lymphocytes (CTL) but are not required for the maintenance of CTL memory following virus infection. J. Virol. 72:7440.[Abstract/Free Full Text]
  48. Thomsen, A. R., A. Nansen, J. P. Christensen, S. O. Andreasen, O. Marker. 1998. CD40 ligand is pivotal to efficient control of virus replication in mice infected with lymphocytic choriomeningitis virus. J. Immunol. 161:4583.[Abstract/Free Full Text]
  49. Vlahos, C. J., W. F. Matter, K. Y. Hui, R. F. Brown. 1994. A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J. Biol. Chem. 269:5241.[Abstract/Free Full Text]
  50. Alessi, D. R., A. Cuenda, P. Cohen, D. T. Dudley, A. R. Saltiel. 1995. PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo. J. Biol. Chem. 270:27489.[Abstract/Free Full Text]
  51. Cuenda, A., J. Rouse, Y. N. Doza, R. Meier, P. Cohen, T. F. Gallagher, P. R. Young, J. C. Lee. 1995. SB 203580 is a specific inhibitor of a MAP kinase homologue which is stimulated by cellular stresses and interleukin-1. FEBS Lett. 364:229.[Medline]
  52. Morel, Y., A. Truneh, R. W. Sweet, D. Olive, R. T. Costello. 2001. TheTNF superfamily members LIGHT and CD154 (CD40 ligand) costimulate induction of dendritic cell maturation and elicit specific CTL activity. J. Immunol. 167:2479.[Abstract/Free Full Text]
  53. Wong, B. R., R. Josien, S. Y. Lee, B. Sauter, H. L. Li, R. M. Steinman, Y. Choi. 1997. TRANCE (tumor necrosis factor [TNF]-related activation-induced cytokine), a new TNF family member predominantly expressed in T cells, is a dendritic cell-specific survival factor. J. Exp. Med. 186:2075.[Abstract/Free Full Text]
  54. Chang, J. T., B. M. Segal, E. M. Shevach. 2000. Role of costimulation in the induction of the IL-12/IL-12 receptor pathway and the development of autoimmunity. J. Immunol. 164:100.[Abstract/Free Full Text]
  55. Leal, I. S., B. Smedegard, P. Andersen, R. Appelberg. 1999. Interleukin-6 and interleukin-12 participate in induction of a type 1 protective T-cell response during vaccination with a tuberculosis subunit vaccine. Infect. Immun. 67:5747.[Abstract/Free Full Text]
  56. Scholler, N., M. Hayden-Ledbetter, A. Dahlin, I. Hellstrom, K. E. Hellstrom, J. A. Ledbetter. 2002. Cutting edge: CD83 regulates the development of cellular immunity. J. Immunol. 168:2599.[Abstract/Free Full Text]
  57. Fukao, T., M. Tanabe, Y. Terauchi, T. Ota, S. Matsuda, T. Asano, T. Kadowaki, T. Takeuchi, S. Koyasu. 2002. PI3K-mediated negative feedback regulation of IL-12 production in DCs. Nat. Immunol. 3:875.[Medline]
  58. Parry, R. V., C. A. Rumbley, L. H. Vandenberghe, C. H. June, J. L. Riley. 2003. CD28 and inducible costimulatory protein Src homology 2 binding domains show distinct regulation of phosphatidylinositol 3-kinase, Bcl-xL, and IL-2 expression in primary human CD4 T lymphocytes. J. Immunol. 171:166.[Abstract/Free Full Text]
  59. Hunt, J. A., F. Kallashi, R. D. Ruzek, P. J. Sinclair, I. Ita, S. X. McCormick, J. V. Pivnichny, C. E. Hop, S. Kumar, Z. Wang, et al 2003. p38 Inhibitors: piperidine- and 4-aminopiperidine-substituted naphthyridinones, quinolinones, and dihydroquinazolinones. Bioorg. Med. Chem. Lett. 13:467.[Medline]
  60. Regan, J., S. Breitfelder, P. Cirillo, T. Gilmore, A. G. Graham, E. Hickey, B. Klaus, J. Madwed, M. Moriak, N. Moss, et al 2002. Pyrazole urea-based inhibitors of p38 MAP kinase: from lead compound to clinical candidate. J. Med. Chem. 45:2994.[Medline]



This article has been cited by other articles:


Home page
 BloodHome page
M. M. Hipp, N. Hilf, S. Walter, D. Werth, K. M. Brauer, M. P. Radsak, T. Weinschenk, H. Singh-Jasuja, and P. Brossart
Sorafenib, but not sunitinib, affects function of dendritic cells and induction of primary immune responses
Blood, June 15, 2008; 111(12): 5610 - 5620.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
E. Jasny, M. Eisenblatter, K. Matz-Rensing, K. Tenner-Racz, M. Tenbusch, A. Schrod, C. Stahl-Hennig, V. Moos, T. Schneider, P. Racz, et al.
IL-12-Impaired and IL-12-Secreting Dendritic Cells Produce IL-23 upon CD154 Restimulation
J. Immunol., May 15, 2008; 180(10): 6629 - 6639.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
T. Kawasaki, M. A. Choudhry, M. G. Schwacha, S. Fujimi, J. A. Lederer, K. I. Bland, and I. H. Chaudry
Trauma-hemorrhage inhibits splenic dendritic cell proinflammatory cytokine production via a mitogen-activated protein kinase process
Am J Physiol Cell Physiol, March 1, 2008; 294(3): C754 - C764.
[Abstract] [Full Text] [PDF]

Home page
 JEMHome page
D. Yang, Q. Chen, S. B. Su, P. Zhang, K. Kurosaka, R. R. Caspi, S. M. Michalek, H. F. Rosenberg, N. Zhang, and J. J. Oppenheim
Eosinophil-derived neurotoxin acts as an alarmin to activate the TLR2-MyD88 signal pathway in dendritic cells and enhances Th2 immune responses
J. Exp. Med., January 21, 2008; 205(1): 79 - 90.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
D. Martinez, M. Vermeulen, E. von Euw, J. Sabatte, J. Maggini, A. Ceballos, A. Trevani, K. Nahmod, G. Salamone, M. Barrio, et al.
Extracellular Acidosis Triggers the Maturation of Human Dendritic Cells and the Production of IL-12
J. Immunol., August 1, 2007; 179(3): 1950 - 1959.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
D. K. R. Karaolis, T. K. Means, D. Yang, M. Takahashi, T. Yoshimura, E. Muraille, D. Philpott, J. T. Schroeder, M. Hyodo, Y. Hayakawa, et al.
Bacterial c-di-GMP Is an Immunostimulatory Molecule
J. Immunol., February 15, 2007; 178(4): 2171 - 2181.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
P. Sen, M. A. Wallet, Z. Yi, Y. Huang, M. Henderson, C. E. Mathews, H. S. Earp, G. Matsushima, A. S. Baldwin Jr, and R. M. Tisch
Apoptotic cells induce Mer tyrosine kinase-dependent blockade of NF-{kappa}B activation in dendritic cells
Blood, January 15, 2007; 109(2): 653 - 660.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
S. Agaugue, L. Perrin-Cocon, F. Coutant, P. Andre, and V. Lotteau
1-Methyl-Tryptophan Can Interfere with TLR Signaling in Dendritic Cells Independently of IDO Activity
J. Immunol., August 15, 2006; 177(4): 2061 - 2071.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
E. Caparros, P. Munoz, E. Sierra-Filardi, D. Serrano-Gomez, A. Puig-Kroger, J. L. Rodriguez-Fernandez, M. Mellado, J. Sancho, M. Zubiaur, and A. L. Corbi
DC-SIGN ligation on dendritic cells results in ERK and PI3K activation and modulates cytokine production
Blood, May 15, 2006; 107(10): 3950 - 3958.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
T. L. Herrmann, C. T. Morita, K. Lee, and D. J. Kusner
Calmodulin kinase II regulates the maturation and antigen presentation of human dendritic cells
J. Leukoc. Biol., December 1, 2005; 78(6): 1397 - 1407.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
Q. Li, A. C. Grover, E. J. Donald, A. Carr, J. Yu, J. Whitfield, M. Nelson, N. Takeshita, and A. E. Chang
Simultaneous Targeting of CD3 on T Cells and CD40 on B or Dendritic Cells Augments the Antitumor Reactivity of Tumor-Primed Lymph Node Cells
J. Immunol., August 1, 2005; 175(3): 1424 - 1432.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
H. K. Kang, H.-Y. Lee, M.-K. Kim, K. S. Park, Y. M. Park, J.-Y. Kwak, and Y.-S. Bae
The Synthetic Peptide Trp-Lys-Tyr-Met-Val-D-Met Inhibits Human Monocyte-Derived Dendritic Cell Maturation via Formyl Peptide Receptor and Formyl Peptide Receptor-Like 2
J. Immunol., July 15, 2005; 175(2): 685 - 692.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
H. Xu, H. Kurihara, T. Ito, H. Kikuchi, K. Yoshida, H. Yamanokuchi, and A. Asari
The Keratan Sulfate Disaccharide Gal(6S03) {beta}1,4-GlcNAc(6S03) Modulates Interleukin 12 Production by Macrophages in Murine Thy-1 Type Autoimmune Disease
J. Biol. Chem., May 27, 2005; 280(21): 20879 - 20886.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
Y. Wang, T. Whittall, E. McGowan, J. Younson, C. Kelly, L. A. Bergmeier, M. Singh, and T. Lehner
Identification of Stimulating and Inhibitory Epitopes within the Heat Shock Protein 70 Molecule That Modulate Cytokine Production and Maturation of Dendritic Cells
J. Immunol., March 15, 2005; 174(6): 3306 - 3316.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yu, Q.
Right arrow Articles by Ostrowski, M. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yu, Q.
Right arrow Articles by Ostrowski, M. A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS