TY - JOUR
AU1 - Wesa, Amy K.
AU2 - Galy, Anne
AB - Abstract The cytokine IL-12, a product of dendritic cells (DC), plays a major role in cellular immunity, notably by inducing lymphocytes to produce IFN-γ. Microbial products, T cell signals and cytokines induce the production of IL-12. Here, IL-1β is identified as a new IL-12-inducing agent, acting conjointly with CD40 ligand (CD40L) on human monocyte-derived DC in vitro. The effects of IL-1β were dose dependent, specifically blocked by neutralizing antibodies, and were observed both in immature and mature DC. Immature DC secreted more IL-12 than mature DC, but the effects of IL-1β were not due to a block of DC maturation as determined by analysis of DC surface markers. The mechanisms of action of IL-1β could be contrasted to that of other inducers of IL-12 such as IFN-γ and lipopolysaccharide (LPS). Either IL-1β or IFN-γ co-induced IL-12 with CD40L but conjointly, IL-1β, CD40L and IFN-γ synergized, inducing very high levels of IL-12. The effects of IL-1β differed from those of LPS in that IL-1β, unlike LPS, could not induce IL-12 solely after IFN-γ priming; and when combined with CD40L, IL-1β, unlike LPS, induced little IL-10. The mechanism of action of IL-1β involves IL-12α mRNA up-regulation, and we show that the combination of CD40L and IL-1β induces high levels of IL-12α and IL-12β mRNA in DC. Altogether, these results delineate a new mechanism linking adaptive and innate immune responses for the regulation of IL-12 production in DC and for the role of IL-1β in the development of cellular immunity. CD40 ligand, cytokines, dendritic cells, human, IL-1, IL-12, IL-10, lipopolysaccharide CD40L CD40 ligand, DC dendritic cells, G-CSF granulocyte colony stimulating factor, GM-CSF granulocyte macrophage colony stimulating factor, IL-12β IL-12β subunit (previously IL-12 p40), IL-12α IL-12α subunit (previously IL-12 p35), LPS lipopolysaccharide, PE phycoerythrin, RPA RNase protection assay, TNF tumor necrosis factor Introduction The cytokine IL-12 is a pro-inflammatory agent that can be used as a potent vaccine adjuvant in infectious disease and cancer models due to its ability to stimulate cellular immunity (reviewed in 1). Targeted or natural mutations occurring in IL-12 or in the components of its receptor have underscored the importance of this cytokine in the control of IFN-γ production by T and NK cells, in the development of Th1 T cell responses, and in the resistance to infections by intracellular organisms (2–4). Phagocytes and antigen-presenting cells, including dendritic cells (DC), produce IL-12 (5). As DC form intimate contacts with lymphocytes, notably with naive T cells, the mechanisms that regulate IL-12 production in DC are central to the study of lymphocyte activation and T cell development. The pro-inflammatory IL-12 molecule is a secreted disulfide linked heterodimer of 70 kDa comprising an α and a β subunit (previously referred to as p35 and p40 chains respectively). Separate mechanisms regulate the production of the two IL-12 chains as well as the secretion of the IL-12 heterodimer both at the transcriptional and post-translational levels, indicating that IL-12 production is tightly controlled and susceptible to regulation by multiple factors (6–8). The production of IL-12 is induced by microbial signals such as lipopolysaccharide (LPS) or CpG bacterial DNA motifs (9,10), by activated T cell signals mediated by CD40–CD40 ligand (CD40L) (11), and further regulated by cytokines such as IFN-γ, IL-4 and granulocyte macrophage colony stimulating factor (GM-CSF) (12,13). While bacterial products are able to induce IL-12 secretion on their own, stimulation with CD40L is not very effective unless provided in conjunction with IFN-γ (14). Recent studies suggest that cross-linking of CD40 be accompanied by a microbial priming signal to result in the secretion of detectable levels of IL-12 in vivo (15). This underscores the requirements for accessory signals allowing IL-12 production in the context of T cell–DC interactions. Some of our preliminary experiments showed that DC produced in, or exposed, to a specific cocktail of cytokines produced more IL-12β in response to CD40L than mature DC, suggesting that some of these cytokines could play an accessory function with CD40L. DC produced by culture of hematopoietic progenitor cells in the presence of flt3 ligand, c-kit ligand, GM-CSF, IL-1β and IL-7 (FKGm17) (16) produced more IL-12β after stimulation with CD40L and induced higher levels of IFN-γ production by T cells in a mixed lymphocyte reaction than mature DC generated from the same hematopoietic progenitor cells in the presence of flt3 ligand, c-kit ligand, GM-CSF, tumor necrosis factor (TNF)-α and IL-4 (FKGmT4) (17–19). IL-1β was tested as a possible candidate molecule that could induce IL-12 due to potential analogy of signaling pathways between members of the IL-1 receptor and the toll-like receptor superfamily. Ligands for toll-like receptors, such as LPS and bacterial lipoproteins, have been reported to induce IL-12 in DC (14,20). Results show that IL-1β is a major inducer of IL-12β production and of IL-12 αβ heterodimer secretion in DC in conjunction with CD40L, and that the effects of IL-1β differ from those of LPS and synergize with IFN-γ to induce very high amounts of IL-12 secretion. Methods Source of cells Human blood samples were obtained with approval from the Institutional Review Board of Wayne State University. Blood from normal adult volunteers was obtained from buffy coats prepared at the American Red Cross (Detroit, MI). Granulocyte colony stimulating factor (G-CSF)-mobilized peripheral blood was obtained from breast cancer patients at the Karmanos Cancer Institute. 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 Adherent monocytes were obtained by incubating MNC on tissue culture plates (2×106 cells/ml/well in 24-well plates) in RPMI medium with 10% FBS (R10) (18) in a humidified atmosphere for 2 h. After removing non-adherent cells by gentle washes, adherent monocytes were induced to differentiate into DC by incubation at 37°C, 5 % CO2, in the presence of several combinations of cytokines and R10 medium. Cytokines included GM-CSF (Immunex, Seattle, WA), TNF-α (RDI, Flanders, NJ; 50 U/ml), IL-4 (kind gift from Dr H. Yssel, DNAX Research Institute, Palo Alto, CA; 100 U/ml) and IL-1β (R & D systems, Minneapolis, MN; or NCI, Biological Resource Branch Repository, Rockville, MD; various concentrations). Activation of DC with cytokines or CD40L After 4–10 days of culture in the presence of indicated cytokines, cells were harvested and washed twice in cytokine-free medium, prior to incubation at the concentration of 2×105 to 106 DC/ml of R10 medium with indicated stimuli such as: IL-1β (various concentrations), human recombinant CD40L (Immunex; 1 μg/ml), LPS (Sigma, St Louis MO; 100 ng/ml) and IFN-γ (R & D Systems; 100 ng/ml). For measures of intracellular contents of IL-12, monensin (Sigma; 2 μM) was added to the DC culture for the last 5 h. For measuring IL-12 secretion by ELISA, supernatants were collected and stored frozen at –20°C until tested. Endotoxin levels in working concentrations of cytokines, media and CD40L were determined to be <0.01 ng/ml by Limulus amoebocyte lysate gel assay (Sigma). Immuno-staining and flow cytometric analysis For cell surface antigen detection, cells were washed in staining buffer (PBS, 0.2% BSA, 0.02% sodium azide) and incubated for 10 min on ice with excess human γ-globulin (1 mg/ml in staining buffer; Gamimune, Miles, Eckhert, IL) to block non-specific Fc binding prior to incubation with directly conjugated mAb such as CD83–phycoerythrin (PE) (Coulter-Immunotech), HLA-DR–FITC, CD54–PE (Caltag) and CD86–FITC (BD-PharMingen). After two washes in staining buffer, propidium iodide (Sigma; 5 μg/ml) was added to permit exclusion of dead cells by flow cytometry. For intracellular staining, cells were washed in PBS then fixed for 5 min in 4% formaldehyde, washed twice in PBS and permeabilized by one wash in a 0.1% saponin (Sigma) solution in staining buffer. Non-specific Fc binding was blocked by incubation for 10 min on ice with 1 mg/ml Gamimune in saponin buffer, then cells were incubated for 30 min on ice with mAb diluted in saponin buffer followed by two washes in saponin buffer. Cells were resuspended in staining buffer prior to analysis. FITC-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-IL-10 (BD-PharMingen) was used for measuring intracellular IL-10. Irrelevant control mAb 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. ELISA Cytokines were measured in supernatants using the OptEIA ELISA kit for IL-12p70 and OptEIA ELISA set for IL-10 according to the manufacturer's instructions (BD-PharMingen). The lower limit of detection for each was 4 pg/ml. Blocking with neutralizing antibodies For blocking experiments, neutralizing antibodies were added at the start of stimulation with CD40L or IL-1β (10 ng/ml). Goat anti-human neutralizing IgG specific for IL-1β and IFN-γ (R & D Systems) were used at 1 and 25 μg/ml respectively for blocking. Normal goat IgG (Jackson ImmunoResearch, West Grove, PA) was used at 25 μg/ml as a control. Non-isotopic RNase protection assay (RPA) Total RNA was isolated using RNA Isolator (Sigma-Genosys, The Woodlands, TX) from day 6 DC treated with and without CD40L (1 μg/ml) and IL-1β (10 ng/ml) for 6–8 h. Biotin-labeled RNA probes were transcribed from PharMingen's Multi-Probe template set using the Maxiscript transcription kit (Ambion, Austin, TX) with T7 RNA polymerase and biotin-14-CTP (Gibco/BRL, Rockville, MD) used at a 40:60 ratio with unlabeled CTP. RPA were performed using Ambion's RPA III kit according to the manufacturer's instructions. Briefly, sample RNA was hybridized to 4 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 an electroblotter, UV cross-linked (Stratagene, La Jolla, CA), then subjected to chemiluminescent detection using Ambion's BrightStar Biodetect kit with CDP-Star substrate. Image analysis was performed using the Kodak Image station 440 CF and 1D Image Analysis software. Results are expressed as the ratio of signal intensity for IL-12 β mRNA levels relative to mRNA levels for the L32 gene. Relative quantitative RT-PCR RNA prepared from DC as described above was incubated with MMLV reverse transcriptase (Promega, Madison, WI) and random hexanucleotide primers for 1 h at 37°C. The resulting cDNA was amplified by PCR using AmpliTaq DNA polymerase [Applied Biosystems (PE Corp), Foster City, CA] and a multiplex PCR kit with primers for IL-12α and 18S ribosomal RNA (Ambion) for 25 cycles (1 min 95°C, 2 min 57°C, 1 min 70°C). Competitive inhibitors to 18S RNA were included at a 4:1 ratio of competimers:primers. PCR products were separated on a 2% agarose gel, stained with ethidium bromide and images were analyzed on the Kodak Image Station. Levels of IL-12α mRNA were expressed as ratio of signal intensity relative to 18S RNA. Results Effects of IL-1β on the induction of IL-12 production in DC Preliminary results showed that progenitor cell-derived DC treated with cytokine combinations containing IL-1β or with IL-1β alone produced more IL-12 in response to CD40L than other preparations of DC (data not shown). This prompted us to test the effects of IL-1β on the regulation of IL-12 in vitro using monocyte-derived DC, since the regulation of IL-12 has been extensively studied in these cells (11,14,21). Immature and mature DC were prepared by culture of adherent monocytes respectively in the presence of GM-CSF and IL-4 or in the presence of cytokine cocktails containing TNF-α, GM-CSF and IL-4 (18). Both mature and immature cells expressed DC markers such as CD40 and HLA-DR. However, while immature DC did not express detectable CD83 at any time point examined, CD83 was found in 34% of mature DC by day 4. Mature and immature DC were subsequently activated for 24 h with IL-1β and/or CD40L to measure IL-12 production. The induction of intracellular IL-12β was measured by flow cytometry. In either type of DC, IL-12β was at background levels without CD40L stimulation but was induced after CD40L stimulation (Table 1). In mature DC, statistically higher IL-12β was obtained after stimulation with CD40L and IL-1β (10 ng/ml) compared to CD40L alone in spite of tissue variations, commonly observed with human samples (Table 1). In immature DC IL-1β had no significant effect on levels of IL-12β at the dose tested of 10 ng/ml. The secretion of the heterodimer IL-12αβ, representing the biologically active pro-inflammatory form of IL-12, was measured by ELISA in supernatant fluid. Under the experimental conditions, stimulation with CD40L alone induced low, albeit detectable, levels of IL-12 heterodimer in either type of DC. Levels were higher in immature DC, thus confirming reports that TNF-α-induced maturation diminished the capacity of DC to secrete IL-12 in response to CD40L (21). Addition of IL-1β to CD40L produced a significant increase in IL-12 secretion, of ~10-fold in mature DC and ~14-fold in immature DC over that induced by CD40L alone. Treatment with IL-1β alone, in the absence of CD40L, did not induce IL-12 secretion. While most of these experiments were performed with DC derived from G-CSF-mobilized peripheral blood, we also confirmed these same effects of IL-1β with DC derived from normal peripheral blood. These results show that IL-1β has a direct effect on DC, and works in conjunction with CD40L to up-regulate the production of IL-12β and the secretion of the IL-12 heterodimer. Dose-dependent effects of IL-1β on immature DC Using DC cultured for periods of 4–10 days, we determined that immature DC produced by culture of monocytes with GM-CSF and IL-4 for 6 days produced the highest levels of IL-12 in response to CD40L (data not shown). Therefore, these cells were used to test the effects of various concentrations of IL-1β on the intracellular production and secretion of IL-12. High concentrations of IL-1β (>30 ng/ml) were required to increase IL-12β levels above those induced by CD40L alone (Fig. 1), thus confirming that a concentration of 10 ng/ml such as used in Table 1 would have no additional effect on IL-12β levels induced by CD40L in immature DC. In contrast, lower concentrations of IL-1β controlled secretion of the heterodimer, with a dose-dependent effect initiated >0.3 ng/ml and plateauing at 30 ng/ml. Thus IL-1β has a dose-dependent effect on CD40L-induced production of IL-12 in immature DC but different doses of IL-1β regulate the production of IL-12β and the secretion of the heterodimer. IL-1β shortens induction time for IL-12 and is required for maximal IL-12 heterodimer secretion The dissociation observed between the effects of IL-1β on intracellular and secreted levels of IL-12 prompted us to analyze the kinetics of production of these two molecular forms. Using day 6 immature DC, we measured IL-12 production at 8, 16, 24, 48 and 72 h after stimulation with IL-1β and/or CD40L. In response to CD40L stimulation alone, DC produced intracellular levels of IL-12β which peaked on or after 24 h post-stimulation (Fig. 2). We detected very little IL-12 heterodimer in the supernatant after CD40L stimulation in agreement with data presented in Table 1. Addition of IL-1β to CD40L treatment caused a shift in IL-12β accumulation, which peaked earlier, at 16 h post-stimulation. A sharp rise in secreted IL-12 heterodimer was detected at 24 h (42.2 pg/1000 cells), with peak production at 48 h post-stimulation (105.9 pg/1000 cells) that persisted for at least 72 h (86.2 pg/1000 cells). Thus, IL-1β accelerated the induction of IL-12 β chain and dramatically augmented the secretion of bioactive IL-12 heterodimer. Maturation effects of IL-1β The immature state of DC coincides with the high production of IL-12 therefore we tested the possibility that IL-1β might induce IL-12 by impairing DC maturation. Maturation state was evaluated by the expression of cell surface markers such as CD54 (ICAM-1) and CD86 which are up-regulated on DC during the CD40L-induced maturation process (Fig. 3). Results showed that IL-1β treatment induced higher levels of these molecules on DC and did not inhibit the maturation induced by CD40L, thus confirming reports that IL-1β is a maturation factor for DC (22). In all experimental conditions tested here, DC expressed equivalent and high levels of cell surface HLA-DR (Fig. 3) and MHC class I antigens (data not shown). The functional maturation state was confirmed in mixed lymphocyte assays showing that DC treated with IL-1β with or without CD40L induced higher T cell proliferation than immature DC (data not shown). We conclude that the mechanism for inducing high levels of IL-12 by IL-1β is not due to an inhibition of maturation. Effects of IL-1β are distinct from those of IFN-γ and are specifically blocked by neutralizing antibodies IFN-γ is a co-inducer of IL-12 that is known to up-regulate the transcription of the IL-12α and IL-12β genes in human blood MNC in response to LPS (6,23). IFN-γ also allows CD40L to induce IL-12 secretion (14). This prompted us to compare the effects of IFN-γ with those of IL-1β and to examine the effects of combining these two cytokines with CD40L. Based on the analysis of three separate blood sample donors, we confirmed that co-treatment of DC with CD40L and either IL-1β or IFN-γ induced IL-12 heterodimer secretion, whereas treatment with CD40L alone did not (Fig. 4). We note that IL-1β or of IFN-γ were used at optimal concentration (data not shown for IFN-γ), indicating similar amplitude of potency for these two cytokines. When used in combination, CD40L, IFN-γ and IL-1β synergized to induce high levels of IL-12 which were ~7-fold greater than levels induced by CD40L with optimal doses of either IFN-γ or IL-1β. The effects of IL-1β were specific. Neutralizing antibodies to IL-1β, but not irrelevant antibodies, abolished IL-12 secretion when added to cultures of DC stimulated with IL-1β and CD40L (Fig. 5). In the same experiment, anti-IFN-γ antibodies were unable to block the secretion of IL-12 thus showing that the effects of IL-1β and CD40L were not indirectly caused by IFN-γ which can be produced by some DC (24). Thus, IL-1β has direct and specific effects on DC, and its mechanism of action is distinct from that of IFN-γ, with which it synergizes to induce even higher levels of IL-12 secretion. Differential effects of LPS and IL-1β on DC cytokines LPS, which is known to regulate IL-12 production, is a common laboratory contaminant, but measurements of endotoxin levels in cytokines and culture media suggested that the effects of IL-1β were not due to the presence of LPS. In fact, LPS had biological activities distinct from IL-1β in our system. Immature DC were treated for 24 h with LPS (100 ng/ml), CD40L and IL-1β in various combinations, and IL-12 was measured. Neither LPS nor IL-1β used as single agents induced IL-12 production but the association of these two stimuli induced the production of IL-12β and the secretion of IL-12 heterodimer in small amounts (Fig. 6). In the presence of CD40L, IL-12β was produced, but the heterodimer was not secreted unless LPS or IL-1β was included and together they had an additive effect on CD40L-induced secretion of IL-12. These flow cytometric data were confirmed by an alternate method, using an ELISA to detect IL-12β production in culture medium (data not shown). A major difference between IL-1β and LPS was found in the regulation of IL-10. Treatment of DC with LPS with CD40L and IL-1β induced the production of substantial numbers of cells producing IL-10, whereas treatment of DC with IL-1β and CD40L did not (Fig. 6). ELISA measurements confirmed these findings, showing in a separate experiment that IL-10 was abundantly secreted by cells treated with CD40L, IL-1β and LPS (64.4 ± 0.4 pg/1000 cells), whereas cells treated with CD40L and IL-1β produced very little IL-10 (7.8 ± 0.2 pg/1000 cells). To determine the cellular source of IL-12 and IL-10, we performed a correlated two-color flow cytometric analysis. Results indicated that IL-10–PE labeled cells were distinct from IL-12–FITC-labeled cells (Fig. 6D). This suggests that monocyte-derived DC populations are not functionally homogeneous and that certain stimuli, CD40L with LPS in this case, may prompt antagonistic functions among subsets of DC. The effects of IL-1β and LPS were further compared in the presence of IFN-γ, which is known to increase the production of IL-12 in response to LPS (14,25). Prior studies have shown that IFN-γ `primes' human blood mononuclear cells to respond to LPS and that a pre-incubation of ~24 h is optimal to induce the secretion of high levels of IL-12 in response to LPS (6). We herein primed immature DC with IFN-γ overnight and then stimulated these DC with various stimuli. Un-primed DC were included for comparison, and in such cells, secretion of IL-12 was not induced by CD40L, but required CD40L and IL-1β co-treatment (Fig. 7). When primed with IFN-γ, IL-12 was produced and secreted in response to either CD40L or LPS. Unlike LPS, IL-1β alone was not able to induce IL-12β production or IL-12α/β secretion after IFN-γ priming (Fig. 7A and B). CD40L and IL-1β synergized with IFN-γ priming to induce high levels of IL-12. IFN-γ priming and treatment with CD40L plus IL-1β induced 80 pg IL-12/1000 cells, which appeared to be 2 times higher than when the three stimuli were used contemporaneously (~40 pg/1000 cells in Fig. 4). The addition of anti-IFN-γ antibodies at the time of priming abolished the effect of IFN-γ on both IL-12β production and IL-12 secretion (Fig. 7A and B). Blocking IFN-γ also shows that the effects of CD40L and IL-1β do not require the presence of endogenous IFN-γ. Blocking IL-1β slightly diminished the amounts of IL-12 induced by LPS suggesting that endogenous IL-1β may be partially involved in the response to LPS. Endogenous IL-1β was not required for CD40L-induced secretion after IFN-γ priming. Altogether, our results show that IL-1β and LPS act as co-inducers of IL-12 with CD40L but that their mechanisms of action are distinct. They differ based on IL-10 induction and responses to IFN-γ priming. The stimulation of DC with IL-1β and CD40L constitutes a powerful signal that induces the production of high levels of IL-12α/β with little IL-10. It synergizes with IFN-γ, leading to the secretion of very high levels of IL-12. Effect of CD40L and IL-1β on IL-12 gene expression Production of IL-12 heterodimer is dependent on the expression of two separate genes. Levels of IL-12α mRNA appear to be constitutively present in many cells, whereas IL-12β mRNA is inducible. In blood MNC and monocytes, IL-12 β and IL-12 α mRNA are up-regulated by LPS, and further increased by LPS and IFN-γ (6,23). DC populations have been shown to up-regulate IL-12 α and IL-12 β mRNAs in response to CD40L stimulation (26). We used an RPA to determine if IL-1β had an effect on IL-12β mRNA levels. Levels of IL-12β mRNA were undetectable in the absence of stimulation or in DC stimulated with IL-1β alone (two experiments, data not shown). DC treated with CD40L alone expressed IL-12β mRNA levels that were detectable in one out of three experiments (Fig. 8A). However, DC treated with the combination of CD40L and IL-1β expressed high levels of IL-12β mRNA in three out of three experiments. Multiplex RT-PCR was used to measure levels of IL-12α mRNA relative to 18S RNA using specific primers that interfere with 18S RNA amplification beyond the range of linearity. Results show that IL-12α mRNA levels are very low in unstimulated cells but are induced by IL-1β or CD40L, or by the combination of CD40L and IL-1β (Fig. 8B). These results are representative of two separate experiments. The observed induction of IL-12α mRNA by IL-1β alone was confirmed in an additional third experiment. Thus, treatment of DC with the combination of CD40L and IL-1β induced high mRNA levels for both IL-12α and IL-12β. These results show that the mechanism for the induction of IL-12 secretion by these agents involves the separate regulation of IL-12 mRNAs, with IL-1β preferentially up-regulating IL-12α mRNA and CD40L preferentially up-regulating IL-12β mRNA. Discussion The present study identifies IL-1β as an inducer of IL-12 secretion in DC in response to CD40L and thus provides a new example for the regulation of IL-12 by signaling through members of the IL-1-receptor/toll-like receptor superfamily (9,14,20). The biological activities of IL-1β were distinguished from those of LPS, a ligand for toll-like receptor 4 by the following observations: unlike LPS, IL-1β required CD40L to induce IL-12 secretion and IL-1β was unable to induce IL-12 solely in response to IFN-γ priming; unlike LPS, IL-1β did not induce high levels of IL-10. This suggests that differential signaling through various members of the IL-1 receptor /toll-like receptor superfamily might be involved in the differential and complex regulation of immune responses. The regulation of IL-12 production is largely at the transcriptional level (reviewed in 1). IL-1β accelerated the accumulation of IL-12β in DC, and this was also observed after LPS and IFN-γ simulation (data not shown), a signal known to increase the transcription of the IL-12α and IL-12β genes (6,27). Indeed, we show that CD40L and IL-1β increased the levels of IL-12α and IL-12β mRNA in DC, and most likely this constitutes the main mechanism responsible for the dramatic effect of IL-1β and CD40L on IL-12 secretion. Most likely IL-1β induces transcriptional activity but it is also possible that IL-1β could act by preventing the degradation of IL-12 mRNA, rather than inducing higher transcriptional activity, as this is a mechanism of action of IL-1 that has described in other hematopoietic cells (28). Our results show for the first time that IL-1β alone can induce IL-12α mRNA levels and confirm that IL-12 secretion may be limited by availability of the IL-12α subunit of the heterodimer. Our results predict that IL-1β may work as a co-stimulation agent for IL-12 secretion with agents inducing IL-12β mRNA. Indeed, LPS which can up-regulate IL-12β mRNA on its own, can be combined with IL-1β to induce the secretion of mild levels of IL-12 (Fig. 6B). Our results also predict that additive or synergistic effects might be obtained between IL-1β and other regulators of IL-12. We observed additive effects between LPS and IL-1β with CD40L. This could be due to additive stimulation of common intracellular signaling molecules including MyD88, IRAK and TRAF6 that activate NF-κB, and thus IL-12 transcription (29). This could also activate separate but convergent signaling pathways, since IL-1β activates MAPK and PI3 kinase pathways, which may synergize with NF-κB to regulate gene expression (reviewed in 30). Our results support the notion that additional signals are required to induce high levels of IL-12 secretion in response to CD40L stimulation (14,15). This appears to contrast with previous studies indicating that CD40L alone could induce IL-12 heterodimer secretion (11,21,31). One explanation for this discrepancy may come from the use of CD40L-transfected cell lines in these studies. Small amounts of IL-1β, a cytokine ubiquitously produced, particularly by stromal cells, may be secreted by such cell lines and small amounts could, based on our results, effectively augment the secretion of bioactive IL-12. It is also possible that CD40L in transfected cells provides a stronger stimulus as a membrane-bound form than as a soluble form. Altogether, our results show that multiple signals may provide more intensity or specificity in determining the response of DC to particular maturation stimuli. Our study is consistent with the general concept that DC integrate multiple types of signals to regulate the production of high levels of bioactive IL-12. The up-regulation of IL-12 provides a new mechanism to delineate the role of IL-1β in inflammatory and immune responses. IL-1β is a ubiquitous cytokine that is released from cells after cleavage of a pro-form by caspase 1 in response to many pathogens. IL-1β activates many different cell types and produces a range of inflammatory activities as it is often involved in innate immune responses (reviewed in 32). Mice deficient in IL-1RI, the receptor that transmits the biological responses of IL-1α and IL-1β, are very susceptible to Listeria infection (33). The ability to mount an immune response against high doses of Listeria requires IL-12 (34) suggesting that IL-1β may be required in a biologically relevant manner to produce IL-12 to control some infections. Levels of IL-1β can be elevated in infectious conditions. In man, plasma levels of IL-1β of as high as 0.5 ng/ml have been reported in severe sepsis (32), which are in the range of concentrations with activity in our in vitro system, as small amounts (0.3 ng/ml) of IL-1β synergized with CD40L to induce IL-12 secretion by DC. Thus, during innate immune responses and/or inflammation, release of IL-1β could act in combination with microbial signals or perhaps with platelet-derived CD40L (35) to induce IL-12 which could be further up-regulated by specific T cell immune responses generating IFN-γ and CD40L on activated T cells. As with many pro-inflammatory mediators, IL-1β, IL-12 and IFN-γ may function in a possible autocrine amplification loop since all of these cytokines can be produced by monocyte-derived DC in response to IL-12 (24). The role of IL-1β in the control of T cell development has been controversial, but studies of IL-1RI-deficient animals suggest that IL-1 proteins may be important in the normal control of Th1/Th2 responses against certain antigens (36). In response to keyhole limpet hemocyanin, IL-1RI-deficient animals produce less of the Th1-associated IFN-γ and more of Th2-associated IL-4 cytokines. It is now understood that the control of T cell development is a multi-factorial event controlled in part by respective levels of polarizing cytokines such as IL-12 and IL-10 in antigen-presenting cells (37). Thus DC stimulated by IL-1β and CD40L that produce high IL-12 with little IL-10 may polarize more strongly toward Th1 T cell development than DC stimulated with LPS which produce more IL-10 in conjunction with IL-12. Altogether, our results delineate a new mechanism for the role of IL-1β in innate and adaptive immunity through the up-regulation of IL-12 secretion by DC. Our results provide an additional link between innate and adaptive immunity in the control of cellular immune responses and further emphasize the importance of environmental instruction in determining the function of DC. Table 1. Effect of IL-1β on IL-12 production     Intracellular IL-12β  Secretion of IL-12 heterodimer    Stimulationb  n  Average cells containing IL-12β (%)c  n  Average IL-12 (pg/1000 cells)d  aImmature DC were generated in Gm/IL-4 while mature DC were generated in the presence of TNF-α.  bDC were washed twice prior to 24 hours with CD40L (1 μg/ml) or IL-1b (10 ng/ml) as indicated.  cIL-12 heterodimer was measured in supernatants by ELISA.  dIL-12β was measured by flow cytometry.  *P<0.05, **P<0.01, ***P<0.0001, NS = not significant by Signed Rank Test.  Immature DCa                  Medium  7  0.02 ± 0.05    13  0.147 ± 0.35  **    IL-1β  5  0.32 ± 0.34    10  0.057 ± 0.12      CD40L  16  4.93 ± 5.59  NS  13  0.293 ± 0.36  ***    CD40L + IL-1β  16  6.44 ± 4.29    13  4.247 ± 4.35    Mature DCa                  Medium  8  0.15 ± 0.32    3  0.008 ± 0.01      IL-1β  8  0.19 ± 0.18    3  0.008 ± 0.01      CD40L  11  3.12 ± 4.87  **  7  0.056 ± 0.01  *    CD40L + IL-1β  11  6.23 ± 8.42    7  0.669 ± 0.99        Intracellular IL-12β  Secretion of IL-12 heterodimer    Stimulationb  n  Average cells containing IL-12β (%)c  n  Average IL-12 (pg/1000 cells)d  aImmature DC were generated in Gm/IL-4 while mature DC were generated in the presence of TNF-α.  bDC were washed twice prior to 24 hours with CD40L (1 μg/ml) or IL-1b (10 ng/ml) as indicated.  cIL-12 heterodimer was measured in supernatants by ELISA.  dIL-12β was measured by flow cytometry.  *P<0.05, **P<0.01, ***P<0.0001, NS = not significant by Signed Rank Test.  Immature DCa                  Medium  7  0.02 ± 0.05    13  0.147 ± 0.35  **    IL-1β  5  0.32 ± 0.34    10  0.057 ± 0.12      CD40L  16  4.93 ± 5.59  NS  13  0.293 ± 0.36  ***    CD40L + IL-1β  16  6.44 ± 4.29    13  4.247 ± 4.35    Mature DCa                  Medium  8  0.15 ± 0.32    3  0.008 ± 0.01      IL-1β  8  0.19 ± 0.18    3  0.008 ± 0.01      CD40L  11  3.12 ± 4.87  **  7  0.056 ± 0.01  *    CD40L + IL-1β  11  6.23 ± 8.42    7  0.669 ± 0.99    View Large Fig. 1. View largeDownload slide Dose-dependent effect of IL-1β on secretion of IL-12 heterodimer. Immature human DC generated by culturing adherent monocytes in GM-CSF and IL-1 for 6 days were treated with CD40L (1 μg/ml) for 24 h in the presence of different concentrations of IL-1β (0–100 ng/ml). Monensin was added for the last 5 h of incubation. Intracellular IL-12 was detected by flow cytometry and is represented as percentage of cells producing IL-12β (shaded bars, scale on left axis). Levels of secreted IL-12 heterodimer were detected in supernatants using ELISA and are shown in pg/1000 cells (dots with connecting line, scale on right axis). Fig. 1. View largeDownload slide Dose-dependent effect of IL-1β on secretion of IL-12 heterodimer. Immature human DC generated by culturing adherent monocytes in GM-CSF and IL-1 for 6 days were treated with CD40L (1 μg/ml) for 24 h in the presence of different concentrations of IL-1β (0–100 ng/ml). Monensin was added for the last 5 h of incubation. Intracellular IL-12 was detected by flow cytometry and is represented as percentage of cells producing IL-12β (shaded bars, scale on left axis). Levels of secreted IL-12 heterodimer were detected in supernatants using ELISA and are shown in pg/1000 cells (dots with connecting line, scale on right axis). Fig. 2. View largeDownload slide Kinetics of IL-12 production is modulated by IL-1β. Immature DC, prepared as in Fig. 1, were treated with CD40L, with and without IL-1β (10 ng/ml), and monensin was added for the last 5 h of the indicated culture times, after which cells and supernatants were harvested for detection of IL-12. Intracellular IL-12 is shown in bars (scale on left axis); IL-12 heterodimer secretion is shown using dots (scale on right axis). IL-1β-treated cultures are shown as shaded bars and black triangles, while cultures not treated with IL-1β are shown as open bars and open circles. One representative experiment of two with identical results is shown. Fig. 2. View largeDownload slide Kinetics of IL-12 production is modulated by IL-1β. Immature DC, prepared as in Fig. 1, were treated with CD40L, with and without IL-1β (10 ng/ml), and monensin was added for the last 5 h of the indicated culture times, after which cells and supernatants were harvested for detection of IL-12. Intracellular IL-12 is shown in bars (scale on left axis); IL-12 heterodimer secretion is shown using dots (scale on right axis). IL-1β-treated cultures are shown as shaded bars and black triangles, while cultures not treated with IL-1β are shown as open bars and open circles. One representative experiment of two with identical results is shown. Fig. 3. View largeDownload slide Effect of CD40L and IL-1β on DC maturation. Cultures of immature DC treated with or without CD40L and IL-1β (10 ng/ml) for 24 h were labeled with PE-conjugated anti-CD54, FITC-conjugated anti-HLA-DR or FITC-conjugated anti-CD86 mAb followed by flow cytometry. One representative experiment of two with identical results is shown as the percentage of cells bound with mAb to surface marker minus the percentage of cells bound with irrelevant control mAb. Fig. 3. View largeDownload slide Effect of CD40L and IL-1β on DC maturation. Cultures of immature DC treated with or without CD40L and IL-1β (10 ng/ml) for 24 h were labeled with PE-conjugated anti-CD54, FITC-conjugated anti-HLA-DR or FITC-conjugated anti-CD86 mAb followed by flow cytometry. One representative experiment of two with identical results is shown as the percentage of cells bound with mAb to surface marker minus the percentage of cells bound with irrelevant control mAb. Fig. 4. View largeDownload slide IL-1β synergizes with CD40L and IFN-γ for high levels of IL-12 secretion. Immature DC prepared as in Fig. 1 were co-treated with CD40L, IL-1β (10 ng/ml) and IFN-γ (100 ng/ml) for 24 h, and the levels of IL-12 heterodimer were measured in the supernatant by ELISA. Results are shown as the average of three independent experiments with different tissues and error bars represent SD. Fig. 4. View largeDownload slide IL-1β synergizes with CD40L and IFN-γ for high levels of IL-12 secretion. Immature DC prepared as in Fig. 1 were co-treated with CD40L, IL-1β (10 ng/ml) and IFN-γ (100 ng/ml) for 24 h, and the levels of IL-12 heterodimer were measured in the supernatant by ELISA. Results are shown as the average of three independent experiments with different tissues and error bars represent SD. Fig. 5. View largeDownload slide Effect of IL-1β on CD40L-induced IL-12 production is specifically inhibited by IL-1β antibody and is independent of IFN-γ. Immature DC prepared as in Fig. 1 were treated with CD40L and IL-1β (10 ng/ml) in the presence of neutralizing antibodies to IFN-γ (25 μg/ml), IL-1β (1 μg/ml) or irrelevant control IgG (25 μg/ml). Supernatants harvested at 18 h were analyzed for IL-12 heterodimer by ELISA. Fig. 5. View largeDownload slide Effect of IL-1β on CD40L-induced IL-12 production is specifically inhibited by IL-1β antibody and is independent of IFN-γ. Immature DC prepared as in Fig. 1 were treated with CD40L and IL-1β (10 ng/ml) in the presence of neutralizing antibodies to IFN-γ (25 μg/ml), IL-1β (1 μg/ml) or irrelevant control IgG (25 μg/ml). Supernatants harvested at 18 h were analyzed for IL-12 heterodimer by ELISA. Fig. 6. View largeDownload slide Differential effect of LPS on DC production of IL-12 and IL-10. Immature DC prepared as in Fig. 1 were treated with CD40L, IL-1β (10 ng/ml) and LPS (100 ng/ml) for 24 h. Intracellular IL-12β (A) was measured by flow cytometry, secreted IL-12 heterodimer was measured with ELISA (B) and intracellular IL-10 (C) was measured by flow cytometry. Results are represented as an average of triplicate wells, with error bars indicating the SD. One representative experiment of two with the same results is shown. (D) Representative dot-plots of DC labeled with IL-12-FITC and IL-10-PE after 24 h in the presence of CD40L, with and without IL-1β and/or LPS. Fig. 6. View largeDownload slide Differential effect of LPS on DC production of IL-12 and IL-10. Immature DC prepared as in Fig. 1 were treated with CD40L, IL-1β (10 ng/ml) and LPS (100 ng/ml) for 24 h. Intracellular IL-12β (A) was measured by flow cytometry, secreted IL-12 heterodimer was measured with ELISA (B) and intracellular IL-10 (C) was measured by flow cytometry. Results are represented as an average of triplicate wells, with error bars indicating the SD. One representative experiment of two with the same results is shown. (D) Representative dot-plots of DC labeled with IL-12-FITC and IL-10-PE after 24 h in the presence of CD40L, with and without IL-1β and/or LPS. Fig. 7. View largeDownload slide Effect of IL-1β on IL-12 production by DC primed with IFN-γ. Immature DC prepared as in Fig. 1 were treated with and without IFN-γ (100ng/ml); those treated with IFN-γ were in the presence of an isotype control (25 μg/ml), anti-IFN-γ (25 μg/ml) or anti-IL-1β (1 μg/ml) neutralizing antibodies. After 16 h, various stimuli were added (1 μg/ml CD40L, 10 ng/ml IL-1β, 100 ng/ml LPS or medium) for an additional 24 h. (A) Cells containing intracellular IL-12β were analyzed by flow cytometry and results represent percentage of cells from pooled triplicate wells. (B) Secretion of IL-12 was measured by ELISA and results represent the average of triplicate wells ± SD. One representative experiment of two independent experiments with similar results is shown. Fig. 7. View largeDownload slide Effect of IL-1β on IL-12 production by DC primed with IFN-γ. Immature DC prepared as in Fig. 1 were treated with and without IFN-γ (100ng/ml); those treated with IFN-γ were in the presence of an isotype control (25 μg/ml), anti-IFN-γ (25 μg/ml) or anti-IL-1β (1 μg/ml) neutralizing antibodies. After 16 h, various stimuli were added (1 μg/ml CD40L, 10 ng/ml IL-1β, 100 ng/ml LPS or medium) for an additional 24 h. (A) Cells containing intracellular IL-12β were analyzed by flow cytometry and results represent percentage of cells from pooled triplicate wells. (B) Secretion of IL-12 was measured by ELISA and results represent the average of triplicate wells ± SD. One representative experiment of two independent experiments with similar results is shown. Fig. 8. View largeDownload slide Effect of IL-1β and CD40L on IL-12 gene expression. Immature DC were treated with and without CD40L (1 μg/ml) and IL-1β (10 ng/ml) for 6 (experiments 1 and 2) to 8 (experiment 3) h and RNA was extracted. (A) Non-isotopic RPA results of three experiments are showed to compare CD40L and CD40L + IL-1β stimulation. Results are represented as the relative expression of IL-12β mRNA normalized to L32 mRNA (housekeeping gene). (B) Multiplex competitive RT-PCR results show IL-12α mRNA levels measured relative to 18S ribosomal RNA as an internal standard. One experiment of two with similar results is shown. Fig. 8. View largeDownload slide Effect of IL-1β and CD40L on IL-12 gene expression. Immature DC were treated with and without CD40L (1 μg/ml) and IL-1β (10 ng/ml) for 6 (experiments 1 and 2) to 8 (experiment 3) h and RNA was extracted. (A) Non-isotopic RPA results of three experiments are showed to compare CD40L and CD40L + IL-1β stimulation. Results are represented as the relative expression of IL-12β mRNA normalized to L32 mRNA (housekeeping gene). (B) Multiplex competitive RT-PCR results show IL-12α mRNA levels measured relative to 18S ribosomal RNA as an internal standard. 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TI - IL-1β induces dendritic cells to produce IL-12
JF - International Immunology
DO - 10.1093/intimm/13.8.1053
DA - 2001-08-01
UR - https://www.deepdyve.com/lp/oxford-university-press/il-1-induces-dendritic-cells-to-produce-il-12-1X2cdWLUpF
SP - 1053
EP - 1061
VL - 13
IS - 8
DP - DeepDyve
ER -