Increased production of pro-inflammatory cytokines and enhanced T cell responses after activation of human dendritic cells with IL-1 and CD40 ligand
© Wesa and Galy; licensee BioMed Central Ltd. 2002
Received: 20 June 2002
Accepted: 18 October 2002
Published: 18 October 2002
Various microbial, inflammatory and immune signals regulate the activation of dendritic cells (DC), determining their ability to interact with naïve T cells and to produce cytokines that direct T cell development. In particular, CD40L and IL-1 cooperatively activate DC to secrete high levels of IL-12. The immuno-stimulatory capacity of such DC is otherwise not well-defined prompting further characterization of the effects of IL-1 and family members on DC activation in comparison with other pro-inflammatory stimuli.
Human DC co-activated in vitro by CD40L and IL-1β expressed numerous cytokine genes including IL-12β, IL-23 p19, IL-1β, IL-1α, IL-1Ra, IL-10, IL-6, IL-18 and IFN-γ. These DC produced high levels of IL-12 protein and appeared capable of producing IFN-γ. Potent CD4+ and CD8+ T cell-stimulatory properties were acquired by DC under conditions that also induced IL-12. Notably, these DC induced rapid differentiation of fluMP-specific CD8+ T cells. Molecules related to IL-1β, like IL-1α, co-induced IL-12 secretion whereas IL-18 did not. Conversely, the inhibitor IL-1Ra, produced endogenously by DC curtailed IL-12 production in response to CD40L.
IL-1 and IL-1Ra play a biologically-relevant role in the positive and negative regulation of DC activation. In conjunction with CD40L, IL-1 sends a powerful activation signal to DC that could be distinguished from other modes of activation. This signal enables the production of pro-inflammatory cytokines by DC, and enhances the differentiation of naïve T cells into effectors of type-1 cellular immune responses.
KeywordsIL-1 CD40L IL-12 IL-23 dendritic cells human
Dendritic cells (DC) are highly effective antigen presenting cells (APCs) capable of stimulating the differentiation of naïve lymphocytes into effector cells (reviewed in ). Specific properties of the DC determine the quality of immune responses that they initiate. These properties are determined by complex and dynamic changes both in the DC and in its environment. Notably, DC undergo a so-called process of maturation that changes migratory properties, diminishes antigen uptake, enhances antigen processing and presentation and induces the production of an array of cytokines (reviewed in ). The qualitative, quantitative and temporal regulation of cytokines produced by DC is thought to be a determining factor in the development of immune responses initiated by these APCs but molecules and mechanisms that regulate such process are not entirely understood.
Activated T cells provide strong stimuli that activate cytokine production in DC notably via interactions between CD40L and CD40 [3, 4]. This interaction not only regulates the production of Th1 polarizing cytokines such as IL-12 by DC in vitro but is a central event in vivo that enables DC to prime effective MHC class I-restricted CD8+ T cell responses [5–7]. IL-12 is a pivotal factor for the initiation of cellular immunity (reviewed in ). IL-12 promotes the polarization of Th1 T cell development, increases the differentiation and activation of cytolytic T cells and in concert with IL-18, induces IFN-γ production by T cells and NK cells. The mechanisms regulating IL-12 production are complex as IL-12 is composed of two chains IL-12α (IL-12p35) and IL-12β (IL-12p40) with independent transcriptional regulation. The two chains assemble as a disulfide-linked heterodimer which is glycosylated and secreted as a biologically active pro-inflammatory and immune mediator [9, 10]. CD40L is known to induce the production of high levels of IL-12β in human monocyte-derived DC . Yet, signalling through CD40 alone is not sufficient to induce the secretion of substantial amounts of IL-12αβ heterodimer. We and others have shown that a second signal, provided by a microbial product (lipopolysaccharide=LPS) or by immune mediators such as IFN-γ or IL-1β, is required to induce the secretion of high levels of IL-12 by DC in the presence of CD40L [12–15].
IL-1 is major pro-inflammatory cytokine with multiple activities in the regulation of immune, inflammatory, endocrine and neuronal systems (reviewed in ). The IL-1 family comprises several structurally-related ligands and the most extensively characterized include IL-1α, IL-1β, the inhibitory IL-1 receptor antagonist (IL-1Ra) and IL-18 (IL-1γ). These ligands bind to members of a family of receptors characterized by immunoglobulin folds in the extracellular portion and Toll/IL-1R (TIR) motif in the intra-cytoplasmic portion . Whereas ligands of Toll-like receptors such as LPS and other microbial products have recently gained a lot of attention as regulators of immune responses in general (reviewed by ) and of DC activation in particular , ligands of IL-1R family have not been as extensively examined under this angle. On its own, IL-1 is known to be a weak maturing agent for DC . However, we have recently shown that when combined with CD40L, IL-1β sends a powerful cooperative signal to monocyte-derived DC, inducing the secretion of high levels of IL-12 . This was recently confirmed and extended to show that IL-1β and CD40L also induce the secretion of high levels of IL-12 in CD34+ cell-derived DC  and A. Wesa, unpublished observation), and that this mode of activation also triggers the production of high levels of IL-6 and the production of IL-1. The effects of IL-1 can be explained at least at the transcriptional level, as CD40L up-regulates IL-12β mRNA and IL-1β complements this effect by up-regulating IL-12α mRNA . Our own results suggest that CD40L co-activation with IL-1β yields high amounts of IL-12 but little IL-10 whereas LPS induces IL-12 but also more IL-10 . As DC encounter various innate, microbial or immune stimuli that engage distinct intracellular signaling pathways, it seems likely that various combinations of these modes of activation might not produce equivalent APCs. The immuno-stimulatory properties of DC activated with IL-1 and C40L have not been well-defined and it is not clear that these cells which produce high amounts of IL-12 will be able to prime and activate naive T cells, in particular CD8+ T cells. This prompted the present study to better characterize the effects of IL-1 and to better understand the molecular regulation of DC function. We found that CD40L+IL-1β stimulation effectively confers immuno-stimulatory activity and could be distinguished from other modes of pro-inflammatory activation of DC. We also found that the constitutive expression of IL-1Ra in DC constitutes a regulatory mechanism curtailing DC activation in the absence of specific inflammatory signals suggesting that the family of IL-1 molecules has biological relevance to DC activation.
Regulation of cytokine gene expression in DC
Regulation of IL-12 and IL-23 secretion in DC
Production of IFN-γ by DC
Regulation of cytokine production by molecules related to IL-1
Both unstimulated DC and activated DC constitutively expressed IL-1Ra mRNA. This IL-1 family member negatively regulates IL-1 by competitively binding to IL-1RI without inducing the signaling cascade (reviewed in ). This suggested that IL-1Ra could perturb the effects of IL-1 signaling in DC and could impact on the regulation of IL-12 production. Figure 5-B shows a representative experiment with the specific inhibition of IL-1Ra by neutralizing antibodies. Anti-IL-1Ra Ab significantly increased the secretion of IL-12 induced by CD40L relative to DC treated with control IgG and CD40L (4.65 ± 3.7 and 0.85 ± 0.7 respectively; p < 0.02, n = 3). The neutralizing anti-IL-1Ra antibody did not affect the production of IL-12 when DC were activated with medium or IL-1β alone which is consistent with the fact that these conditions do not up-regulate IL-12β on their own. The neutralizing antibody also did not affect the levels of IL-12 produced in response to CD40L+IL-1β. In this case, the exogenous IL-1 provided maximal induction of IL-12 and was probably largely in excess of the endogenous IL-1Ra, therefore inhibition of IL-1Ra did not provide additional induction of IL-12. Altogether, these results provide evidence that both IL-1Ra mRNA and IL-1 mRNA are translated in CD40L-treated DC, and act in a biologically relevant manner. Further, this demonstrates that constitutive IL-1Ra expression regulates the response of DC to endogenous IL-1.
DC stimulated by CD40L+IL-1β effectively prime and stimulate the differentiation of CD4+ and CD8+ T cells
Effects of DC activation on priming of CD8+ T cell responses
DC used in priming (b)
Percent of CD8+ cells binding Flu-MP-HLA-A2 tetramer
Absolute number of Flu-MP specific cells
Fold expansion Flu-MP specific T cells
CD40L + IL-1β
CD40L + IL-1β
CD40L + IL-1β
CD40L + IL-1β
CD40L + IL-1β
We herein show that various pro-inflammatory molecules can generate distinct activation profiles in DC and that members of the IL-1 family of proteins play a biologically relevant role in the regulation of the functional differentiation of DC.
Multiple activation signals appear to be required to induce the production of high levels of cytokines by DC. The production of IL-12 which is a pivotal event in the development of type-1 cellular immunity is effectively induced in vitro by treating DC with CD40L+IL-1β and even more so by CD40L+IL-1β+IFN-γ, in agreement with recent findings by Luft et al. . Such synergistic concept is consistent with in vivo studies showing the dependence on both microbial signals and endogenous CD40L to induce effective immunity . Exposure to multiple pro-inflammatory mediators is likely to occur under biological circumstances as these mediators can be transcriptionally induced in a contemporary manner and are regulated by autocrine loops, commonly described in inflammation . In combination with CD40L, IL-1 induces the production of IL-12, IL-1, IFN-γ and IL-18 in DC. IL-18, acting in combination with IL-12 could induce large amounts of IFN-γ by lymphocytes, further sensitizing DC to produce extremely high levels of IL-12. The role played by DC-derived IFN-γ remains unclear. Neutralization studies suggest that IFN-γ is not responsible for IL-12 production in response to IL-1β and CD40L . As IFN-γ is produced approximately 24 hours after stimulation it seems that its role would be to contribute to T cell activation rather than to DC activation itself. DC-derived IFN-γ has been reported in the mouse , albeit under distinct activating conditions. Based on these studies it remains to be tested if IFN-γ production by human DC might also be indirectly due to the presence of IL-12 and IL-18.
Here, we report distinct pro-inflammatory stimuli that all induce high levels of IL-12 but appear to provide otherwise distinct responses. In contrast to IL-1, co-stimulation with IFN-γ induced relatively little IL-10, IL-6, IL-18 and IFN-γ. LPS induced higher IL-10, higher IL-1Ra and strikingly less IL-23. The difference in the ability to produce IL-10 was confirmed at the protein level (data not shown and ). While IL-12 is important for naïve CD4+ T cell priming, IL-23 is reportedly important for memory CD4+ cells . Differences between IL-12 and IL-23 production suggest that DC may display distinct cytokine profiles that correspond to distinct phases of an immune response. Another interpretation is that DC may interpret distinct pathogen signals, for instance IL-1 or LPS, to mount different types of cytokine reponses [19, 25, 26]. This would be desirable to optimize host defense against particular aggressions or to be effective in particular tissues. The biological consequences of these differences will have to be determined in future studies.
An important aspect of the autocrine regulation of cytokines in DC is the production of negative regulators such as IL1Ra. Genetic invalidation of IL-1Ra not only up-regulates inflammation but also affects the immune system, causing spontaneous development of auto-immune arthritis in certain strains of mice and rendering other strains more susceptible to immunogens causing this disease . The precise mechanisms underlying the role of IL-1Ra in immune responses are complex, but implicate antigen presentation. APCs of mice lacking IL-1Ra induce higher proliferation and higher activation of wild-type CD4+ T cells than control APCs, resulting in the up-regulation of CD40L, OX40 and IL-2Rα, and thus have the opposite effects of APCs lacking IL-1 . Our own data provide a potential mechanistic explanation clearly showing for the first time that IL-1Ra can have a direct role on APC function by regulating the secretion of IL-12. Neutralization experiments in DC showed that IL-1Ra inhibited the secretion of IL-12 in response to CD40L. This is most likely through negative regulation of endogenous IL-1 responses as well as CD40L-induced IL-1 in DC. While the RPA technique did not reveal IL-1 transcripts in unstimulated immature DC, it has been reported that IL-1β mRNA are detected by the more sensitive RT-PCR analysis in freshly-isolated Langerhans cells, generally considered a paradigm for unstimulated immature DC . Furthermore, direct evidence of IL-1β protein secretion has been shown in monocyte-derived DC . This demonstrates that autocrine cytokine regulation via IL-1 family members is a biologically relevant mechanism in DC. As IL-1Ra appears to be a negative regulator of DC activation, it is notable that the LPS+IFN-γ stimulation induces the highest levels of IL-1Ra mRNA in DC. As high levels of IL-1α, IL-1β are also induced, IL-1Ra may provide a negative balance to prevent the establishment of an IL-1-mediated autocrine stimulation cascade and to curtail the effects of LPS. Thus, IL-1Ra may be one significant built-in mechanism in the APC to regulate its immuno-stimulatory properties and to prevent the development of uncontrolled immunity. The biological relevance of these conclusions seems further supported by genetic analyses studies in humans. Polymorphisms in the IL-1Ra gene such as the IL1RN*2 allele cause quantitative differences in both IL-1Ra and IL-1β production and associate with the severity of inflammatory and auto-immune diseases such as ulcerative colitis and Crohn's disease, lupus erythematosus and possibly with coronary artery disease. Conversely, these polymorphisms negatively associate with various infections such as vaginal colonization with mycoplasmas, cytomegalovirus, Epstein-Barr virus, human immunodeficiency virus and with the occurrence of ovarian cancer (recently reviewed in ).
Our results therefore identify IL-1 as an effective co-activator of DC in conjunction with CD40L. This is consistent with the fact that IL-1 has been recognized as an adjuvant for almost 2 decades  and may refine our understanding of the effects of IL-1 in the context of DC-initiated immunity. Thus, IL-1 which is produced locally and systemically in response to inflammation or infection may play an important role as a T cell-independent factor complementing the effects of T cell-derived CD40L. Upon encountering these two molecules, DC would become more effective APCs, producing IL-12, IL-23, IL-18 and IFN-γ and stimulating the differentiation of CD4+ and CD8+ T cells. Besides implications in inflammatory, infectious or auto-immune diseases, our data are also consistent with the identification of IL-1 as an important adjuvant of CTL immunization in vivo in conjunction with IL-12, IL-18 or IFN-γ . Like IL-1, IL-12 is not critically required for the generation of CTL, yet the addition of exogenous IL-12 or the inhibition of IL-12 respectively improve or inhibit the development of CTL [33, 34]. Our results confirm that IL-12 is not essential for priming against fluMP since neutralizing Abs do not block the development of specific T cells. However, we show that enhanced production of IL-12 or IL-23 by CD40L+IL-1-activated DC mediates the enhancing effects on the priming of CD8+ T cell responses.
Altogether, we predict that CD40L+IL-1β-treated DC will function as effective vaccines for the induction of cellular immunity in vivo. As we see that various degrees of pro-inflammatory functional differentiation can be acquired by DC, it seems that the rational use of adjuvants based on molecular regulation of cytokines in DC may yield useful vaccines for immunotherapy.
Source of cells
Human blood samples were obtained with approval from the Institutional Review Board of Wayne State University. G-CSF mobilized peripheral blood was obtained from breast cancer patients at the Karmanos Cancer Institute. Cord blood samples were obtained from Hutzel Hospital, Detroit MI. Mononuclear cells (MNC) were isolated by centrifugation over Ficoll (Amersham Pharmacia Biotech, Piscataway, NJ) (d < 1.077 g/ml) and were cryopreserved in liquid nitrogen using a 10% DMSO freezing solution.
Culture of DC
Monocytes were obtained by incubating MNC on tissue culture plates (2 × 106 cells per ml per well in 24 well plates) in RPMI medium with 10% fetal bovine serum (FBS) (R10)  in a humidified atmosphere at 37°C, 5% CO2 for 2 hours, followed by washing to remove non-adherent cells. These adherent cells were cultured in R10 medium with GM-CSF (25 ng/ml, Immunex, Seattle, WA), and IL-4 (100 U/ml, kind gift from Dr. H Yssel, DNAX Research Institute, Palo Alto, CA) to induce DC differentiation.
Following 6 days of culture, cells were harvested and washed twice in cytokine-free medium, prior to incubation with stimuli such as: human recombinant CD40L trimer (1 μg/ml; kind gift of Immunex), IL-1β (10 ng/ml, unless stated otherwise; Biological Resource Branch Repository, Rockville, MD), LPS (100 ng/ml; Sigma, St. Louis MO), IFN-γ (100 ng/ml; R&D Systems), IL-1α (various concentrations; R&D Systems), IL-18 (various concentrations; R&D Systems), TNF-α (50 ng/m; RDI, Flanders, NJ). To measure intracellular cytokine production, monensin (2 μM; Sigma) was added to the DC culture for the last 5 hours. To measure cytokine secretion by ELISA, supernatants were collected after 24 hours and stored frozen at -20°C until tested. Endotoxin levels in working concentrations of cytokines, media and CD40L were determined to be less than 0.01 ng/ml by Limulus amoebocyte lysate gel assay (Sigma).
Immuno-staining and flow cytometric analysis
Intracellular staining was done as previously described , Briefly, after fixation in 4% formaldehyde, cells were washed twice in PBS then permeabilized in saponin buffer (0.1% saponin (Sigma), 0.2% BSA, 0.02% sodium azide, in PBS). Non-specific Fc binding was blocked by incubation for 10 min. on ice with excess human gamma-globulin in saponin buffer (1 mg/ml; Gamimune, Miles, Eckhert, IL), then cells were incubated for 30 minutes on ice with mAbs diluted in saponin buffer followed by 2 washes in saponin buffer. Cells were resuspended in wash buffer without saponin prior to analysis. FITC or APC-conjugated anti-IL-12 (BD-PharMingen), which recognizes the IL-12 β-chain either alone or in IL-12 α/β heterodimer, was used for detection of intracellular IL-12β in DC, while PE-conjugated anti-IFN-γ (clone 4S.B3, Pharmingen) were used for measuring intracellular IFN-γ by DC or T cells. Irrelevant control mAbs were used as negative controls of non-specific staining. Cells were analyzed on a FACSCalibur instrument (Becton Dickinson) and data were analyzed using WinMDI (Version 2.8) software.
IFN-γ was measured in supernatants using the OptEIA ELISA set for IFN-γ according to manufacturer's instructions (BD-PharMingen). The lower limit of detection was 4 pg/ml.
Non-isotopic RNase protection assay (RPA)
Following activation with various stimuli for 8 hours (unless otherwise indicated), total RNA was isolated from DC using RNA Isolator (Sigma-Genosys, The Woodlands, TX). Biotin labeled RNA probes were transcribed from PharMingen's Multi-Probe template set using Maxiscript transcription kit (Ambion Inc., Austin, TX) withT7 RNA polymerase and biotin-14-CTP (Gibco-BRL, Rockville, MD) used at a 40:60 ratio with unlabeled CTP. RPAs were performed using Ambion's RPA III kit according to manufacturer's instructions. Briefly, sample RNA was hybridized to 8 ng biotin-labeled probes overnight at 56°C, then digested with RNase A/T. Digested probes were analyzed on a 8 M urea/5% polyacrylamide gel (19:1acrylamide:bis-acrylamide, (BioRad, Hercules, CA)) in TBE. Separated probes were transferred to a positively charged nylon membrane by a semi-dry electroblotter (Owl Separation Systems, Portsmouth, NH), UV cross-linked (Stratagene, La Jolla, CA), then subjected to chemiluminescent detection using Ambion's BrightStar Biodetect kit with CDP-Star substrate. Membranes were exposed either to Kodak Biomax ML film, or directly to the Kodak Image station 440 CF for a one-hour capture. Images analysis was performed using the Kodak Image station 440 CF and 1D Image Analysis software. Results are expressed as the ratio of net signal intensity for a particular mRNA to the net signal intensity of the L32 housekeeping gene.
Immunoprecipitation and western analysis
DC supernatants were concentrated by centrifugation through YM-10 concentrators (Millipore Corp., Bedford, Massachusetts) then immunoprecipitated with anti-IL-12β (C8.6, PharMingen) and A/G agarose bead conjugates (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Precipitants were separated by 10% SDS-PAGE under non-reducing conditions, transferred to PVDF membrane, and blotted with anti-IL-12p70-specific rat monoclonal Ab 20C2 (PharMingen) or anti-IL-12 goat polyclonal Ab (R&D Systems). HRP-conjugated goat-anti-rat or donkey-anti-goat polyclonal secondary Abs were visualized using the ECL detection system (Amersham Biosciences, Piscataway, NJ). Blots were exposed to Biomax ML film as above.
Blocking with neutralizing antibodies
To examine the effects of IL-1ra on DC activation, goat anti-human neutralizing IgG specific for IL-1Ra (R&D Systems) and normal goat IgG (R&D Systems) were added at the start of DC stimulation with CD40L. To examine the effects of IL-12 in priming experiment 4, polyclonal neutralizing Abs to IL-12 ((R&D Systems) or normal goat IgG (1 μg/ml), were added in the T cell/DC culture at initiation of priming.
Naïve CD8+ T cell priming
DC, prepared from HLA-A2+ cord blood (CB) MNC, were activated overnight as described above, then pulsed with 10 μg/ml influenza matrix peptide Flu-MP (58–66 GILGFVTL; Genemed Synthesis, San Francisco, CA). Autologous purified T cells were obtained by negative selection, using mAbs specific for CD14 (3C10), CD40 (G28.5), CD32 (IV-3), CD11b (OKM1), glycophorin A (10F7MN) (ATCC), and HLA-DP/DQ (SPVL-3) (kind gift of Dr. H Yssel), followed by incubation with sheep anti-mouse magnetic beads (Dynal, Lake Success, NY). Cells were further depleted with mAbs to HLA-DR and CD16 (Caltag) and a second round of selection with beads. The resulting cell preparations contained > 95% CD3+ T cells. Peptide pulsed-DC were irradiated (400-cGy 137Cs source), washed twice, then incubated with purified T cells in R10 medium supplemented with 10 ng/ml IL-7 (R&D Systems) at 37°C, 5% CO2. Cultures were replenished with R10 and IL-7 on day 3–4 by demi-depletion, and were examined for the presence of antigen specific cells by flow cytometry on day 7. Briefly, T cells were counted then incubated in the dark at 37°C for 15 min. with APC-labeled Flu-MP-HLA-A2 tetramer constructs (NIAID Tetramer Facility and the NIH AIDS Research and Reference Reagent Program, Atlanta, GA). After washing, cells were incubated with anti-CD8-FITC (OKT8, ATCC, conjugated in our lab) and anti-CD45RA-PE (Caltag) mAbs, then washed. Cells were resuspended with 5 μg/ml propidium iodide (Sigma) for exclusion of dead cells and analyzed by flow cytometry. The limit of detection of CD8+ Flu-MP-HLA-A2 tetramer+ T cells was 0.02–0.04% as determined by dilutions of a flu-MP specific HLA-A2+ T cell line in HLA-A2- cord blood MNC. Negative control consisted of HLA-A2- cord blood MNC in which < 0.01% (n = 3) CD8+ Flu-MP-HLA-A2 tetramer+ cells were detected. Percent of CD8+ cells binding Flu-MP-HLA-A2 tetramer were calculated by setting quadrants based on staining with isotype control Abs (staining <0.01%), and calculated as (% Flu-MP-HLA-A2 tetramer+ CD8+ cells) / (% total CD8+ cells). Absolute number of tetramer+ cells was calculated as (% CD8+ Flu-MP-HLA-A2 tetramer+ cells) × (total number of cells in culture after priming). Fold expansion specific T cells was calculated as (absolute number of tetramer+ cells after priming) / (absolute number of tetramer+ cells in initiating cultures).
Mixed lymphocyte reaction (MLR)
The procedure has been described elsewhere . Briefly, preparations >90% pure T cells were obtained from MNC by removal of cells recognized by mAbs to CD14 (3C10), CD40 (G28.5), CD32 (IV-3), CD11b (OKM1), glycophorin A (10F7MN), (ATCC, Manassas, VA) HLA-DP/DQ (SPVL-3, kind gift of Dr. H Yssel) and CD16 (KD1, kind gift of Dr. Ferlazzo, Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy), HLA-DR and CD15 (Caltag) using indirect panning on goat-anti-mouse IgG (Sigma)-coated plates, followed by a second round of negative selection with sheep anti-mouse magnetic beads (Dynal, Lake Success, NY). Populations of CD4+ or CD8+ T cells were obtained by removing the opposite subset in the second round of purification. Allogeneic DC were irradiated and cultured with pure T cells at the concentration of 20% DC:T cell ratio in R10 medium for 8 days. On day 8, supernatant fluids were collected to measure cytokine content by ELISA and cells were harvested to be re-stimulated for 5 hours on plates coated with mAbs to CD3 and CD28 (2 μg/ml each) (PharMingen, San Diego, CA). Monensin (2 μM; Sigma) was added to enable the detection of intracellular IFN-γ by flow cytometry.
Total RNA prepared from DC was reverse transcribed after incubation with Superscript II reverse transcriptase (Gibco) and random hexanucleotide primers for one hour at 37°C. The resulting cDNA was amplified by PCR using AmpliTaq DNA polymerase (Applied Biosystems (PE Corp), Foster City, CA) and primers for p19 (forward: 5' AGC AGC TCA AGG ATG GCA CTC AG 3'; reverse: 5' CCC CAA ATT TCC CTT CCC ATC TA 3') and for 18S ribosomal RNA (Ambion) for 35 cycles (1 min 95°C, 2 min 56°C, 1 min 70°C). PCR products were separated on a 2% agarose gel, stained with ethidium bromide.
Paired t-Test analysis was used to assess statistical differences where indicated.
- CD40L = recombinant human CD40 ligand:
DC = dendritic cells, TLR = Toll-like receptor, RPA = RNase protection assay
The authors acknowledge the support of the NIH-NIAID tetramer facility in Atlanta, GA, Immunex in Seattle, WA and Drs Yssel and Ferlazzo for kind gifts of reagents; the flow cytometry facility of the Barbara Ann Karmanos Cancer Institute of Wayne State University for technical support; Dr. Anthony Johnson from the Department of Obstetrics at Wayne State University for procurement of cord blood samples; Drs. Baynes and Klein from the Bone Marrow Transplant Program of the Barbara Ann Karmanos Cancer Institute of Wayne State University for procurement of mobilized peripheral blood and Drs. Wei, Firat, Chouaib and Vainchenker for critical review of the manuscript. This work was supported in part by the American Cancer Society grant RPG-98-183-01, by the National Institutes of Health NCI R01 CA82884-03 grant and by the NCI training grant T32 CA09531 to the Program in Cancer Biology.
- Mellman I, Steinman RM: Dendritic cells: specialized and regulated antigen processing machines. Cell. 2001, 106: 255-258.View ArticlePubMedGoogle Scholar
- Lanzavecchia A, Sallusto F: Regulation of T cell immunity by dendritic cells. Cell. 2001, 106: 263-266.View ArticlePubMedGoogle Scholar
- Caux C, Massacrier C, Vanbervliet B, Dubois B, Van Kooten C, Durand I, Banchereau J: Activation of human dendritic cells through CD40 cross-linking. J Exp Med. 1994, 180: 1263-1272.View ArticlePubMedGoogle Scholar
- Sallusto F, Cella M, Danieli C, Lanzavecchia A: Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: downregulation by cytokines and bacterial products. J Exp Med. 1995, 182: 389-400.View ArticlePubMedGoogle Scholar
- Ridge JP, DiRosa F, Matzinger P: A conditioned dendritic cell can be a temporal bridge between a CD4+ T-helper and a T-killer cell. Nature. 1998, 393: 474-478. 10.1038/30989.View ArticlePubMedGoogle Scholar
- Bennet SRM, Carbone FR, Karamalis F, Flavell RA, Miller JFAP, Heath WR: Help for cytotoxic T-cell response is mediated by CD40 signalling. Nature. 1998, 393: 478-480. 10.1038/30996.View ArticleGoogle Scholar
- Schoenberger SP, Toes REM, van der Voort EIH, Offringa R, Melief CJM: T-cell help for cytotoxic T lymphocytes is mediated by CD40-CD40L interactions. Nature. 1998, 393: 480-483. 10.1038/31002.View ArticlePubMedGoogle Scholar
- Ma X, Trinchieri G: Regulation of interleukin-12 production in antigen-presenting cells. Adv Immunol. 2001, 79: 55-92.View ArticlePubMedGoogle Scholar
- Ma X, Chow JM, Gri G, Carra G, Cerosa F, Wolf S, Dzialo R, Trinchieri G: The interleukin 12 p40 gene promoter is primed by interferon γ gamma in monocytic cells. J Exp Med. 1996, 183: 147-157.View ArticlePubMedGoogle Scholar
- Carra G, Gerosa F, Trinchieri G: Biosynthesis and posttranslational regulation of human IL-12. J Immunol. 2000, 164: 4752-4761.View ArticlePubMedGoogle Scholar
- Cella M, Scheidegger D, Palmer-Lehmann K, Lane P, Lanzavecchia A, Alber G: 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. 1996, 184: 747-752.View ArticlePubMedGoogle Scholar
- Snijders A, Kalinski P, Hilkens CM, Kapsenberg ML: High-level IL-12 production by human dendritic cells requires two signals. Int Immunol. 1998, 10: 1593-1598. 10.1093/intimm/10.11.1593.View ArticlePubMedGoogle Scholar
- Schulz O, Edwards DA, Schito M, Aliberti J, Manickasingham S, Sher A, Reis e Sousa C: CD40 triggering of heterodimeric IL-12 p70 production by dendritic cells in vivo requires a microbial priming signal. Immunity. 2000, 13: 453-462.View ArticlePubMedGoogle Scholar
- Wesa AK, Galy A: IL-1 beta induces dendritic cells to produce IL-12. Int Immunol. 2001, 13: 1053-1061. 10.1093/intimm/13.8.1053.View ArticlePubMedGoogle Scholar
- Luft T, Jefford M, Luetjens P, Hochrein H, Masterman KA, Maliszewski C, Shortman K, Cebon J, Maraskovsky E: IL-1beta enhances CD40 ligand-mediated cytokine secretion by human dendritic cells (DC): a mechanism for T cell-independent DC activation. J Immunol. 2002, 168: 713-722.View ArticlePubMedGoogle Scholar
- Dinarello CA: Interleukin-1, interleukin-1 receptors and interleukin-1 receptor antagonist. Int Rev Immunol. 1998, 16: 457-499.View ArticlePubMedGoogle Scholar
- Rock FL, Hardiman G, Timans JC, Kastelein RA, Bazan JF: A family of human receptors structurally related to Drosophila Toll. Proc Natl Acad Sci U S A. 1998, 95: 588-593. 10.1073/pnas.95.2.588.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhang G, Ghosh S: Toll-like receptor-mediated NF-kappaB activation: a phylogenetically conserved paradigm in innate immunity. J Clin Invest. 2001, 107: 13-19.PubMed CentralView ArticlePubMedGoogle Scholar
- Kadowaki N, Ho S, Antonenko S, Malefyt RW, Kastelein RA, Bazan F, Liu YJ: Subsets of human dendritic cell precursors express different toll-like receptors and respond to different microbial antigens. J Exp Med. 2001, 194: 863-869. 10.1084/jem.194.6.863.PubMed CentralView ArticlePubMedGoogle Scholar
- Oppmann B, Lesley R, Blom B, Timans JC, Xu Y, Hunte B, Vega F, Yu N, Wang J, Singh K, Zonin F, Vaisberg E, Churakova T, Liu M, Gorman D, Wagner J, Zurawski S, Liu Y, Abrams JS, Moore KW, Rennick D, de Waal-Malefyt R, Hannum C, Bazan JF, Kastelein RA: Novel p19 protein engages IL-12p40 to form a cytokine, IL-23, with biological activities similar as well as distinct from IL-12. Immunity. 2000, 13: 715-725.View ArticlePubMedGoogle Scholar
- Hochrein H, Shortman K, Vremec D, Scott B, Hertzog P, O'Keeffe M: Differential production of IL-12, IFN-a, and IFN-g by mouse dendritic cell subsets. J Immunol. 2001, 166: 5448-5455.View ArticlePubMedGoogle Scholar
- Debets R, Timans JC, Homey B, Zurawski S, Sana TR, Lo S, Wagner J, Edwards G, Clifford T, Menon S, Bazan JF, Kastelein RA: Two novel IL-1 family members, IL-1 delta and IL-1 epsilon, function as an antagonist and agonist of NF-kappa B activation through the orphan IL-1 receptor-related protein 2. J Immunol. 2001, 167: 1440-1446.View ArticlePubMedGoogle Scholar
- Arend WP, Malyak M, Guthridge CJ, Gabay C: Interleukin-1 receptor antagonist: role in biology. Annu Rev Immunol. 1998, 16: 27-55. 10.1146/annurev.immunol.16.1.27.View ArticlePubMedGoogle Scholar
- Dinarello CA: Biologic basis for interleukin-1 in disease. Blood. 1996, 87: 2095-2147.PubMedGoogle Scholar
- Hirschfeld M, Weis JJ, Toshchakov V, Salkowski CA, Cody MJ, Ward DC, Qureshi N, Michalek SM, Vogel SN: Signaling by toll-like receptor 2 and 4 agonists results in differential gene expression in murine macrophages. Infect Immun. 2001, 69: 1477-1482. 10.1128/IAI.69.3.1477-1482.2001.PubMed CentralView ArticlePubMedGoogle Scholar
- Huang Q, Liu D, Majewski P, Schulte LC, Korn JM, Young RA, Lander ES, Hacohen N: The Plasticity of Dendritic Cell Responses to Pathogens and Their Components. Science. 2001, 294: 870-875. 10.1126/science.294.5543.870.View ArticlePubMedGoogle Scholar
- Horai R, Saijo S, Tanioka H, Nakae S, Sudo K, Okahara A, Ikuse T, Asano M, Iwakura Y: Development of chronic inflammatory arthropathy resembling rheumatoid arthritis in interleukin 1 receptor antagonist-deficient mice. J Exp Med. 2000, 191: 313-320. 10.1084/jem.191.2.313.PubMed CentralView ArticlePubMedGoogle Scholar
- Nakae S, Asano M, Horai R, Sakaguchi N, Iwakura Y: IL-1 enhances T cell-dependent antibody production through induction of CD40 ligand and OX40 on T cells. J Immunol. 2001, 167: 90-97.View ArticlePubMedGoogle Scholar
- Heufler C, Topar G, Koch F, Trockenbacher B, Kampgen E, Romani N, Schuler G: Cytokine gene expression in murine epidermal cell suspensions: interleukin 1 beta and macrophage inflammatory protein 1 alpha are selectively expressed in Langerhans cells but are differentially regulated in culture. J Exp Med. 1992, 176: 1221-1226.View ArticlePubMedGoogle Scholar
- Witkin SS, Gerber S, Ledger WJ: Influence of interleukin-1 receptor antagonist gene polymorphism on disease. Clin Infect Dis. 2002, 34: 204-209. 10.1086/338261.View ArticlePubMedGoogle Scholar
- Staruch MJ, Wood DD: The adjuvanticity of interleukin 1 in vivo. J Immunol. 1983, 130: 2191-2194.PubMedGoogle Scholar
- Staats HF, Bradney CP, Gwinn WM, Jackson SS, Sempowski GD, Liao HX, Letvin NL, Haynes BF: Cytokine requirements for induction of systemic and mucosal CTL after nasal immunization. J Immunol. 2001, 167: 5386-5394.View ArticlePubMedGoogle Scholar
- Bhardwaj N, Seder RA, Reddy A, Feldman MV: IL-12 in conjunction with dendritic cells enhances antiviral CD8+ CTL responses in vitro. J Clin Invest. 1996, 98: 715-722.PubMed CentralView ArticlePubMedGoogle Scholar
- Chouaib S, Chehimi J, Bani L, Genetet N, Tursz T, Gay F, Trinchieri G, Mami-Chouaib F: Interleukin 12 induces the differentiation of major histocompatibility complex class I-primed cytotoxic T-lymphocyte precursors into allospecific cytotoxic effectors. Proc Natl Acad Sci U S A. 1994, 91: 12659-12663.PubMed CentralView ArticlePubMedGoogle Scholar
- Ferlazzo G, Klein J, Paliard X, Wei W, Galy A: Dendritic cells generated from CD34+ progenitor cells with flt3-ligand, c-kit-ligand, GM-CSF, IL-4 and TNF-a are functional antigen-presenting cells resembling mature monocyte-derived DC. Journal of Immunotherapy. 2000, 23: 45-58. 10.1097/00002371-200001000-00007.View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.