HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Stober, D. Articles by Reimann, J. Search for Related Content PubMed PubMed Citation Articles by Stober, D. Articles by Reimann, J.

IL-12/IL-18-Dependent IFN- Release by Murine Dendritic Cells¹

Department of Medical Microbiology, University of Ulm, Ulm, Germany

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Dendritic cells (DC) develop in GM-CSF-stimulated cultures from murine bone marrow progenitors in serum-free (or low serum) medium. CD11c⁺ myeloid DC from 7-day cultures stimulated with TNF-, IFN-, IFN-, or LPS up-regulated surface expression of CD40 and CD86 costimulator and MHC class II molecules, did not up-regulate the low "spontaneous" release of IL-18, and did not release IFN-. Stimulation of in vitro-generated DC with exogenous IL-12 and IL-18 (but not with IL-4 or LPS plus IL-18) induced IFN- expression and release in 15–20% of the DC (detectable by FACS analyses or ELISA). Endogenous IL-12 p70 produced by DC in response to ligation of CD40 stimulated IFN- release when exogenous IL-18 was supplied. In vivo-generated, splenic CD8⁺ and CD8^- DC (from immunocompetent and immunodeficient H-2^d and H-2^b mice) cultured with IL-12 and IL-18 released IFN-. The presence of LPS during the stimulation of DC with IL-18 plus endogenous (CD40 ligation) or exogenous IL-12 did not affect their IFN- release. In contrast, splenic DC pretreated in vitro or in vivo by LPS strikingly down-regulated IFN- release in response to stimulation by IL-18 and (endogenous or exogenous) IL-12. Hence, DC are a source of early IFN- generated in response to a cascade of cytokine- and/or cell-derived signals that can be positively and negatively regulated.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Specific activation of alternative effector functions in an immune response is known as polarization. T cell priming depends on professional APCs that deliver early polarizing signals to naive T cells (reviewed in Ref. 1). Dendritic cells (DC)³ are the most potent APC known (reviewed in Refs. 2, 3). When immature, tissue-resident DC encounter a pathogen, they capture its Ags in a microenvironment loaded with signals originating from either the pathogen or nonspecific tissue responses to the pathogen. This stimulates their migration and maturation into presentation-competent APC. In lymph nodes (remote from the site of pathogen invasion), DC prime naive T cells, thereby initiating the polarization of the emerging T cell response (1). DC thus seem to be not only key mediators for delivering Ag-specific information to T cells but also convey to them information on the protective effector functions required to eliminate a pathogen. In addition to their role of specifically stimulating T cell responses, the modulation of the Th1- or Th2-promoting capacity of DC has gained considerable interest.

DC of the myeloid lineage (mDC) are potent stimulators of T cell responses. Murine and human bone marrow (BM) precursors develop in vitro into mDC in cultures supplemented with GM-CSF and other cytokines, e.g., IL-4, TNF-, or Flt3 ligand (FL, fms-like tyrosine kinase 3 ligand) (4, 5, 6, 7, 8, 9). DC generated in this system can be used to study the cascade of signals that modulate the phenotype of immature, mature, or activated DC with Th1- or Th2-promoting or -inhibiting capacity. IL-12 is required to stimulate immune responses of IFN--producing CD4⁺ T cells (10, 11, 12, 13, 14) and CD8⁺ CTL (15, 16, 17, 18). This is evident by the deficient Th1 immunity in STAT4^-/-, IL-12^-/-, or IL-12^-/- knockout (KO) mice (19, 20, 21). DC-derived IL-12 plays a key role in priming Th1 T cell responses (reviewed in Refs. 11, 22). A major signal that triggers IL-12 release is the ligation of CD40 (23, 24, 25, 26, 27). Signaling through CD40 induces IL-12 secretion that synergizes with IL-2 for the induction of IFN- production by T cells (23). Signaling of immature DC in vivo is required to induce their competence to respond to CD40 ligation with IL-12 release (28). Th1 polarization has been shown to depend on APC-derived IL-12 and T cell-derived IFN- (12, 29). As T cell-derived IFN- usually appears only 2–4 days after the initiation of a response, an alternative early source for this cytokine would facilitate early polarization of T cell responses. The cytokine IL-18 facilitates priming of Th1 immune responses in synergy with IL-12 (30, 31, 32). IL-18 and IL-12 synergistically stimulate IFN- production by T cells (33, 34, 35), B cells (36), NK cells (37), macrophages (38, 39), and DC (40, 41). Both major regulator and effector cytokines for Th1 immune responses, IL-12 as well as IFN-, can thus be produced by DC. We studied the signals that regulate IFN- production by murine DC either generated in vitro from BM progenitors or generated in vivo and isolated as splenic DC.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

BALB/cJ (H-2^d) mice, C.B-17^+/+ and C.B-17^scid/scid (SCID) (H-2^d) mice, and C57BL/6J (H-2^b) mice were bred and kept under standard pathogen-free conditions in the animal colony of the University of Ulm (Ulm, Germany). Breeding pairs of these mice were obtained from Bomholtgard (Ry, Denmark). BALB/c STAT4^-/- KO mice were provided by M. J. Grusby (Department of Immunology and Infectious Diseases, Harvard School of Public Health and Department of Medicine, Harvard Medical School, Boston, MA) (19). A breeding colony of these mice was established in Ulm. Female mice were used at 10–16 wk of age.

Generation of DC from BM

The in vitro generation of DC from murine BM has been described (4). Briefly, BM cells (BMC) prepared from femurs were depleted of CD4⁺ CD8⁺ B220⁺ lymphocytes and MHC class II⁺ cells (cat. no. 492-01, 494-01, 495-01, 524-01; Miltenyi Biotec, Bergisch Gladbach, Germany) by MACS following the manufacturer’s instructions. These BMC depleted of T cells, B cells, and maturing myeloid cells were cultured at a density of 10⁶ cells/ml (cat. no.150 229; Nunc, Wiesbaden, Germany) in UltraCulture medium (BioWhittaker, Verviers, Belgium) supplemented with 5 ng/ml GM-CSF and 10 ng/ml FL (cat. no. 315-03 and 300-19; PeproTech, Rocky Hill, NJ), 2 mM glutamine, and antibiotics. DC developed from C57BL/6 (B6)-derived BMC in serum-free cultures; growth of DC from BALB/c-derived BM progenitors required 0.5% v/v FCS supplements to the medium. Cultures were incubated at 37°C in humidified air with 5% CO₂. On days 3 and 5, cells were fed by medium exchange.

Flow cytometry analyses of DC

Cells were suspended in PBS/0.3% w/v BSA supplemented with 0.1% w/v sodium azide. Nonspecific binding of Abs to FcR was blocked by preincubating cells with the anti-CD16/CD32 mAb 2.4G2 (1 µg/10⁶ cells; cat. no. 01240D; PharMingen, Hamburg, Germany). Cells were incubated with 0.5 µg/10⁶ cells of the relevant mAb for 30 min at 4°C, washed twice, and subsequently incubated with a second-step reagent for 15 min at 4°C. Cells were washed twice and analyzed on a FACScan (BD Biosciences, Mountain View, CA). Dead cells were excluded by propidium iodide staining. The following reagents and mAbs from PharMingen were used: PE-conjugated anti-I-A^d/I-E^d (cat. no. 06345A); PE-conjugated anti-I-A^b (cat. no. 06045A); biotinylated anti-H-2D^b,d,k (cat. no. 06232D); PE-conjugated anti-CD80 (B7-1) (cat. no. 09605B); PE-conjugated anti-CD40 (cat. no. 09665B); FITC-conjugated anti-CD86 (B7-2) (cat. no. 09215B); PE-conjugated anti-CD11c (cat. no. 09705B); and FITC-conjugated anti-CD54 (ICAM-1) (cat. no. 01544D). PE-conjugated F4/80 was purchased from Cedarlane Laboratories (Hornby, Ontario, Canada) (cat. no. CL 8940PE). Furthermore, we used the rat anti-mouse DEC-205 (NLDC-145) mAb (cat. no. MCA949) from Biozol (Eching, Germany), the FITC-conjugated IgG1 mAb R3-34 (cat. no. 20614A), PE-conjugated IgG1 mAb R3-34 (cat. no. 20615A), and streptavidin-Red670 (cat. no. 19543-024; Life Technologies, Eggenstein, Germany).

IFN- expression of stimulated DC was detected by intracellular staining. Brefeldin A (5 µg/ml) was added for the last 4 h of the incubation period of stimulated DC cultures (4 µl Golgi-Stop/6 ml culture medium; Cytofix/Cytoperm Plus; PharMingen cat. no. 2076KK). Cells were harvested, washed twice in staining buffer (PBS without Mg²⁺/Ca²⁺, 0.3% w/v BSA, 0.1% w/v sodium azide), incubated (15 min, 4°C) with purified 2.4G2 Ab to block nonspecific binding of Ab to FcRs, washed with staining buffer, resuspended in HBSS (cat. no. 24020-091; Life Technologies), surface stained with a conjugated anti-CD11c Ab, washed twice with HBSS, resuspended in 100 µl Cytofix/Cytoperm solution for 20 min at 4°C, and washed twice in 1 ml 1x Perm/Wash solution. Fixed and permeabilized cells were resuspended in 50 µl 1x Perm/Wash solution and stained for 30 min at 4°C with either the FITC-conjugated anti-IFN- Ab (cat. no. 18114A) or FITC-conjugated rat IgG1 isotype control Ab (cat. no. 20614A). Cells were washed twice in 1x Perm/Wash solution and suspended in staining buffer. Cells (10⁴) were analyzed by flow cytometry using a FACScan equipped with a 15-mW argon laser (BD Biosciences) and CellQuest software (BD Biosciences). Analysis gates were set on CD11c⁺ cells.

Splenic DC

Splenic DC were purified as described by Fukao et al. (41). Briefly, spleens were cut into small pieces and incubated for 45 min in RPMI 1640 medium containing collagenase I (0.5 mg/ml) and DNase (0.1 mg/ml). EDTA (at a final concentration of 10 mM) was added for the last 3 min of the incubation period. The tissue pieces were minced through a nylon mesh. Single cells were harvested, washed, resuspended in Nycodenz overlaid with RPMI 1640 medium, and centrifuged at 4°C at 9500 x g for 20 min. Cells on the interface were collected, washed twice, and cultured for 2 h to allow DC to adhere. Nonadherent cells were removed, and adherent DC were incubated overnight to detach. From these cells, contaminating B and T cells were depleted by MACS using biotinylated anti-CD3, anti-CD4, anti-B220 mAb (PharMingen), and streptavidin-coupled microbeads. From the CD3^-CD4^-B220^- population, CD8⁺ DC were positively selected by MACS. From the CD8^- fraction, CD11c⁺ DC were positively selected by MACS. Flow cytometric analyses demonstrated that these DC subsets contained >98% CD8⁺CD11c⁺ cells or CD8^-CD11c⁺ DC. These purified DC were cultured at 5 x 10⁴/well with the indicated amounts of cytokines in RPMI 1640/5% FCS in 150 µl flat-bottom microwells.

LPS injection of mice

Mice were injected i.v. 6 h before sacrifice with either 50 µg LPS (in 200 µl PBS) per mouse (cat. no. L-2143; Sigma, St. Louis, MO) or 200 µl PBS (solvent control).

Cytokines and cytokine detection by ELISA

The following recombinant mouse cytokines were obtained from PeproTech: IL-4 (cat. no. 214-14), IL-18 (cat. no. 315-04), TNF- (cat. no. 315-01A), GM-CSF (cat. no. 315-03), and FL (cat. no. 300-19). IL-12 p40 (cat. no. 499-ML) was purchased from R&D Systems (Wiesbaden, Germany). IFN- (cat. no. 19301T) and IL-12 p70 (cat. no. 19361V) were obtained from PharMingen. Universal type I IFN (human IFN- A/D, BglII) was obtained from PBL Biomedical Laboratories (New Brunswick, NY).

Cytokines in supernatants were detected by conventional double-sandwich ELISA. For detection and capture, the following mAbs (from PharMingen) were used: mAb R4-6A2 (cat. no. 18181D) and biotinylated mAb XMG1.2 (cat. no. 18112D) were used for IFN-, mAb C15.6 (cat. no. 18491D) or mAb RedT/G297-289 (cat. no. 20011D) were used as coating Abs for IL-12 p40 and IL-12 p70 ELISA, respectively; and mAb C17.8 (cat. no. 18482D) was used for detection in both cases. For detection of IL-18, pairs of unconjugated and biotinylated Abs (cat. no. MAB422, BAF422) were purchased from R&D Systems. Extinction was analyzed at 405/490 nm on a TECAN microplate ELISA reader (TECAN, Crailsheim, Germany) using the EasyWin software (TECAN). The detection limits of the cytokine ELISAs were determined to be 10 pg/ml for IL-12 p40, 10 pg/ml for IL-12 p70 and IFN-, and 60 pg/ml for IL-18.

Cytokine stimulation of in vitro-generated DC

On day 7 of culture, nonadherent cells were harvested. CD11c⁺ cells were purified by MACS (cat. no. 520-01; Miltenyi Biotec) and replated in UltraCulture medium supplemented with GM-CSF, FL, glutamine, antibiotics, and in the case of BALB/c-derived DC with 0.5% v/v FCS. In some experiments, DC were cultured for a further 2–3 days in medium to which the indicated cytokines were added at a concentration of 20 ng/ml; LPS was added at 1 µg/ml; and IFN- was added at 500 U/ml. On day 9–10 of culture, CD11c⁺ cells were analyzed by flow cytometry or used in further studies. CD11c⁺ DC were washed three times and seeded into round-bottom microwells at 2 x 10⁵ cells/200 µl in UltraCulture medium with GM-CSF, FCS, and antibiotics. These cultures were stimulated with the indicated amounts of IL-12 p70, IL-18, and/or IL-4. In some experiments, irradiated (18,000 rad) CD40 ligand (CD40L)-transfected J558L cells (or nontransfected J558L control cells) were added at 6.6 x 10⁴/well. After a 48-h incubation period, supernatants were harvested and tested for cytokines by ELISA.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
The generation of mDC from BM progenitors

We generated DC from (T cell-, B cell-, mature myeloid cell-depleted) BM progenitors of BALB/cJ, BALB/c STAT4^-/-, or B6 mice in cultures supplemented with GM-CSF and FL. DC were generated from B6-derived BMC in serum-free cultures; growth of DC from BALB/c-derived BM progenitors required 0.5% v/v FCS supplements to the medium. Of the nonadherent cells harvested from 7-day cultures, 50–70% were CD11c⁺. The CD11c⁺ cells were CD11b⁺NLDC-145⁺CD54⁺CD4^-CD8^- and expressed readily detectable levels of MHC class I molecules and CD80 (Fig. 1 and data not shown). Expression of the macrophage-specific markers CD14 or F4/80 was not detected (data not shown). This phenotype resembles that previously described for immature mDC (42, 43, 44). In CD11c⁺ mDC populations from day 7 cultures, a subset of 20–30% of the cells showed up-regulated surface expression of MHC class II, CD40, and CD86 molecules (Fig. 1). Incubating mDC for another 2 days in GM-CSF and FL increased the fraction of DC with up-regulated surface expression of costimulator and MHC class II molecules (Figs. 1 and 2 and data not shown). Purified mDC (from all mouse strains tested) cultured with GM-CSF/FL "spontaneously" released low amounts of IL-12 p40 and IL-18, but no IFN- into the supernatant (Fig. 3 and data not shown). Thus, the majority of in vitro-cultured mDC maintained an immature phenotype during a 7- to 9-day culture period, but a small subset of these cells spontaneously matured under serum-free culture conditions. In this system, we studied signals that trigger IFN- release by DC.

View larger version (40K): [in this window] [in a new window]   FIGURE 1. Surface phenotype of CD11c+ DC harvested from 7-day BMC cultures. Nonadherent cells were harvested from serum-free day 7 B6 BMC cultures. CD11c+ DC were purified from this cell population by MACS sorting, stained with Abs to CD11c, CD54, CD40, CD80, CD86, I-Ab (MHC class II), Kb (MHC class I), and NLDC-145 (DEC-205), and analyzed by FACScan. Dead cells were excluded by propidium iodide. A representative example of seven independent analyses is shown.

View larger version (33K): [in this window] [in a new window]   FIGURE 2. Surface phenotype of cytokine-stimulated, BM-derived CD11c+ DC. Purified CD11c+ DC were harvested from day 7 BMC cultures and restimulated in vitro for another 2 days (at a density of 106 cells/well) in GM-CSF/FL-containing medium supplemented with no cytokines (none) or 20 ng/ml IL-4, IL-12, IL-18, TNF-, IFN-, 500 U/ml IFN- type 1, or 1 µg/ml LPS. The cell surface phenotype of the DC was analyzed by flow cytometry.

View larger version (14K): [in this window] [in a new window]   FIGURE 3. IL-12 release by cytokine-stimulated CD11c+ DC. CD11c+ DC purified from day 7 BMC cultures were cultured in vitro in GM-CSF/FL-containing medium (at 106 cells/well) for 2 days with 20 ng/ml IL-4, IL-18, TNF-, IFN-, 500 U/ml IFN- type 1, 1 µg/ml LPS, or J558/CD40L transfectants (3.3 x 105/ml). IL-12 p70 and IL-12 p40 were detected in supernatants of DC cultures by conventional double-sandwich ELISA. Mean values of triplicates (±SD) of one of three experiments are shown.

TNF, IFN, and LPS up-regulate MHC class II and costimulator expression by DC, have a limited effect on IL-12 release and no effect on IL-18 release, and do not trigger IFN- release

Cytokines induce DC maturation (5, 45, 46, 47, 48, 49). CD11c⁺ mDC from 7 day cultures were incubated with titrated amounts of cytokines or LPS. TNF-, type I and II IFNs, and LPS (but not IL-4 and IL-18) up-regulated surface expression of MHC class II and costimulator (CD40, CD54, CD80, and CD86) molecules (Fig. 2 and data not shown). Incubation with LPS or TNF- (but not with IL-4, IL-18, type I or II IFNs) enhanced IL-12 p70 and p40 release (Fig. 3). None of the cytokines tested induced IFN- release or up-regulated IL-18 release by mDC (data not shown). These data confirm that TNF-, IFN, and LPS induce DC maturation. These stimuli have only a limited effect on IL-12 p70 release, did not enhance the spontaneous IL-18 release, and did not induce IFN- release by DC.

Synergistic action of IL-12 and IL-18 on DC stimulates IFN- release

IFN- was not released by DC stimulated with cytokines or LPS. In contrast, stimulation with IL-12 plus IL-18 efficiently induced IFN- release by DC (Fig. 4A). This confirms a recent report (41). IL-4 codelivered with either IL-12 or IL-18 (over a broad dose range) did not trigger IFN- release by DC, as evident in four independent experiments using different mouse strains. (Fig. 4, A and B and data not shown). This observation is in contrast to previously reported data (41). IL-12 responsiveness of DC was required to generate an IFN- response after synergistic stimulation with IL-12 and IL-18 because DC from STAT4^-/- KO BALB/c mice did not release IFN- after IL-12/IL-18 stimulation (Fig. 5). Titration experiments showed that fairly high doses of 20–60 ng/ml IL-18 were required to stimulate IFN- release by DC costimulated with 20 ng/ml IL-12 (Fig. 4B). IFN- release by DC stimulated with IL-12 and IL-18 increased substantially over a 3- to 4-day culture period (Fig. 4C). Cytoplasmic staining of DC for IFN- followed by flow cytometry analyses showed that 15–20% of the cells expressed IFN- after a 96-h stimulation with IL-12/IL-18 (Fig. 4D). The IFN-⁺ and IFN-^- mDC subsets showed no detectable difference in surface marker expression. They were CD4^-CD8^- and displayed reduced levels of CD11c, CD11b, and DEC-205 on the cell surface (data not shown). Exogenous IL-12 and IL-18 hence synergistically stimulate expression and release of IFN- by mDC.

View larger version (23K): [in this window] [in a new window]   FIGURE 4. CD11c+ DC release IFN- after stimulation with IL-12 and IL-18. A, Purified CD11c+ DC were stimulated for 72 h with 20 ng/ml IL-12, IL-18, IL-4, or combinations of these cytokines. IFN- was measured by ELISA in supernatants conditioned by stimulated DC. B, IL-18, but not IL-4, induces IFN- release by CD11+ DC in synergy with IL-12 (20 ng/ml). C, Kinetic of IFN- release by CD11c+ DC stimulated with IL 12 (20 ng/ml) and IL-18 (60 ng/ml) for 48–96 h. D, A subset of 15–20% of CD11c+ DC expresses cytoplasmic IFN- after stimulation with IL-12 and IL-18, as detected by flow cytometric analyses.

View larger version (13K): [in this window] [in a new window]   FIGURE 5. IL-12 responsiveness of DC is required for the induction of IFN- release after stimulation with IL-12 and IL-18. DC were generated from BM of either normal BALB/c mice or IL-12-unresponsive STAT4-/- KO BALB/c mice. Purified CD11c+ DC were cultured for 72 h with either no cytokine supplements or with IL-12 and IL-18 (20 ng/ml each). IFN- was measured by ELISA in supernatants conditioned by stimulated DC. Mean values of triplicates (±SD) of one of two independent experiments are shown.

IL-12 p70 released by DC stimulated through CD40 induces IFN- release in synergy with IL-18

As shown in Fig. 3 and in previous reports (23, 24, 25, 27, 50), CD40 ligation is a potent stimulus that triggers IL-12 release by DC. CD40 ligation (coculture with the CD154/CD40L-expressing cell line J558L/CD154) of DC produced 120 ng/ml IL-12 p40 in BALB/c mice and 25 ng/ml IL-12 p40 in B6 mice; in both mouse strains tested CD40 ligation triggered release of similar amounts 0.3–0.5 ng/ml IL-12 p70 (Figs. 3 and 6A). Release of both immunosuppressive IL-12 p40 and bioactive IL-12 p70 is thus substantially increased by CD40 ligation. In BALB/c mice, a 250-fold excess of IL-12 p40 over IL-12 p70 was released whereas in B6 mice this excess was 80-fold (Fig. 6A). This was found in seven independent experiments using BM-derived DC from BALB/c and B6 mice.

View larger version (19K): [in this window] [in a new window]   FIGURE 6. Modulation of IL-12 p40, IL-12 p70, and IFN- release of DC in response to CD40 ligation by cytokines or LPS. A, Purified, BM-derived CD11c+ DC from BALB/c or B6 mice were cocultured in GM-CSF/FL-supplemented medium for 2 days with CD40L-expressing J558L/CD40L transfectants. IL-12 p40 and IL-12 p70 were determined by double-sandwich ELISA. B, Purified, BM-derived CD11c+ DC from B6 mice were cultured in GM-CSF/FL-supplemented medium for 2 days with 20 ng/ml IL-4, IL-12, IL-18, TNF-, or 1 µg/ml LPS. The DC were washed and cocultured for an additional 2 days with CD40L-expressing J558L/CD40L transfectants. IL-12 p40 and IL-12 p70 were determined by double-sandwich ELISA. C, Purified, BM-derived CD11c+ DC from B6 mice were cultured in GM-CSF/FL-supplemented medium for 2 days with either no additional stimuli (pretreatment in vitro: none) or with 1 µg/ml LPS (pretreatment in vitro: LPS). The DC were washed and cultured for an additional 2 days with IL-18 plus IL-12, IL-18 plus the CD40L-expressing J558L/CD40L transfectants, IL-18 plus LPS, or IL-18 plus LPS plus J558L/CD40L transfectants. IFN- was measured by ELISA in supernatants conditioned by stimulated DC. Mean values of triplicates (±SD) of one of four independent experiments are shown.

View larger version (12K): [in this window] [in a new window]   FIGURE 7. CD40 ligation generates endogenous IL-12 that synergizes with IL-18 in stimulating IFN- release by DC. Purified, BM-derived CD11c+ DC from B6 mice were stimulated for 72 h with nontransfected J558L cells (lane 1), 20 ng/ml IL-12 (lane 2), 60 ng/ml IL-18 (lane 3), both IL-12 and IL-18 (lane 4), CD40L-expressing J558L/CD40L transfectants (lane 5), J558L/CD40L cells and IL-18 (lane 6), or J558L/CD40L cells, 20 ng/ml IL-18, and 10 µg/ml anti-IL-12 p35 Ab (lane 7). IFN- was determined in supernatants by double-sandwich ELISA. Mean values of triplicates (±SD) of one of three independent experiments are shown.

Autocrine IL-18 release by DC is insufficient to prime them for IFN- release in response to IL-12

BM-derived DC spontaneously release low levels of IL-18 during culture. Neither the ligation of CD40 on the surface of DC nor the stimulation of DC by LPS or cytokines (TNF-, type I or II IFNs, IL-4, or IL-12) enhanced the spontaneous IL-18 release by DC (data not shown). Furthermore, these cytokines did not enhance the spontaneous IL-18 release by DC stimulated by either LPS or surface CD40 ligation (data not shown). Exogenous IL-18 at a dose >10 ng/ml was required to trigger a detectable IFN- response by mDC (Fig. 4B). The IL-18 release of cultured DC was thus low and not up-regulated by the tested stimuli that induce DC maturation. This level of IL-18 release was insufficient to support autocrine induction of IFN- release by DC. To efficiently stimulate IFN- release by DC under physiological conditions, either an additional (cytokine or cell interaction) trigger is required to enhance IL-18 production by DC or IL-18 has to be supplied by an alternative source.

Modulation of IL-12 p70 release by DC induced by CD40 ligation has little effect on their IFN- response

CD40 ligation stimulates DC (from different inbred mouse strains) to release bioactive IL-12 p70 and immunosuppressive IL-12 p40 (Fig. 6A). DC pretreated with IL-4 or TNF- before CD40 ligation showed enhanced release of IL-12 p70 as well as a 3- to 10-fold reduction of the p40:p70 ratio of the released IL-12 (Fig. 6B). Pretreatment of DC with IL-12, IL-18, or LPS had little effect on their IL-12 p70 release in response to CD40 ligation (Fig. 6B and data not shown). When IL-4, TNF-, or LPS were present during the stimulation of DC by exogenous IL-18 and CD40 ligation, their IFN- release was not modulated (Fig. 6C and data not shown). Hence, these well-characterized maturation signals for DC have only a limited effect on IFN- release costimulated by IL-12/IL-18.

DC pretreated in vitro with LPS down-modulate IFN- release in response to IL-12/IL-18 costimulation

We tested whether in vitro pretreatment of DC with LPS (that induces their maturation) enhances their IFN- response to IL-12/IL-18 stimulation. As evident in four independent experiments, the presence of LPS during the IL-12/IL-18 or CD40 ligation/IL-18 costimulation had no effect on the IFN- release by DC (Fig. 6C and data not shown). In contrast, IFN- release by DC triggered by stimulation with either IL-12/IL-18 or CD40 ligation/IL-18 was down-regulated when DC were pretreated for 48 h with LPS (Fig. 6C). The presence of LPS during stimulation of LPS-pretreated DC with IL-18 or CD40 ligation/IL-18 did not affect their IFN- response (Fig. 6C). Hence, in vitro LPS pretreatment down-regulates IFN- release of DC in response to IL-12/IL-18 stimulation.

Splenic CD8^- and CD8⁺ DC release IFN- in response to IL-12/IL-18 stimulation

We tested whether freshly isolated splenic DC produce IFN- in response to IL-12/IL-18 stimulation. CD8^- and CD8⁺CD11c⁺ DC were isolated from collagenase-digested spleen cell populations from B6 mice, immunocompetent BALB/c, or immunodeficient C.B-17^scid/scid (SCID) mice. CD8⁺ DC are lymphoid-related, whereas a majority of CD8^- DC seem to be myeloid-related (51). The purity of the CD8^- and the CD8⁺ DC subsets was confirmed by flow cytometry and was always found to be >98% for CD11c⁺ MHC class II^high cells. Cells of these DC subsets were cultured in medium with 10 ng/ml GM-CSF supplemented with IL-12 and/or IL-18. B6-derived CD8^- and CD8⁺ DC released IFN- in response to IL-12/IL-18 stimulation (Fig. 8); the IFN- release by lymphoid (CD8⁺) DC was reproducibly higher than that of myeloid (CD8^-) DC, confirming a previous report (40). B6-derived CD8⁺ DC produced IFN- also in response to stimulation with IL-12, without exogenous IL-18 supplements (Fig. 8). Splenic CD8^- and CD8⁺ DC from immunocompetent or immunodeficient BALB/c mice released comparable amounts of IFN- when stimulated with IL-12/IL-18 that were similar to the amounts of IFN- released by B6-derived CD8^- DC (Fig. 8). CD8⁺ DC from BALB/c mice did not release IFN- in response to stimulation with IL-12 alone, i.e., IL-18 was an essential costimulus for the release of IFN- in mice of BALB/c but not B6 background (Fig. 8). This pattern was observed in three independent experiments. When splenic CD8^- or CD8⁺ DC from all tested mouse strains were stimulated with IL-4 and IL-18 or TNF- and IL-18, they did not release IFN- (data not shown). These data are in contrast to reported data (41). BM-derived DC developing from progenitors in vitro, as well as splenic CD8^- or CD8⁺ DC that developed in vivo, thus release IFN- after stimulation with IL-12/IL-18.

View larger version (13K): [in this window] [in a new window]   FIGURE 8. Splenic CD8+ or CD8- DC produce IFN- after IL-12/IL-18 stimulation. CD11c+ DC were isolated from the spleen of immunocompetent BALB/c or B6 mice or severely immunodeficient (SCID) C.B-17scid/scid mice. CD8+ or CD8- CD11c+ DC subsets were prepared as described in Materials and Methods. These cells were cultured in GM-CSF-supplemented medium for 3 days, either with no additional cytokines or with IL-12, IL-18, or IL-12 plus IL-18. IFN- was determined in supernatants by double-sandwich ELISA. Mean values of triplicates (±SD) of one of five independent experiments are shown.

Splenic DC from mice pretreated in vivo with LPS strikingly down-regulate IFN- release in response to IL-12/IL-18

We showed that DC pretreated in vitro with LPS down-regulate IFN- release stimulated by IL-12/IL-18 (Fig. 6C). We tested whether the pretreatment of DC in vivo impairs their ability to produce IFN- when stimulated by IL-12/IL-18.

Spenic DC obtained from LPS-treated mice showed evidence of activation, i.e., up-regulation of CD40, CD86, and MHC class II surface expression (Fig. 9A). Splenic DC from LPS-pretreated mice produced lower amounts of IL-12 p40 and showed almost complete suppression of IL-12 p70 release after CD40 ligation (Fig. 9B). IFN- release by splenic CD11c⁺ DC from LPS-pretreated mice was strikingly reduced after in vitro stimulation by either IL-12/IL-18 or CD40 ligation/IL-18 when compared with IFN- release by splenic DC from nontreated mice (Fig. 9B). This was observed with immunocompetent BALB/c and B6 mice in three independent experiments. The presence of LPS in the in vitro stimulation cultures had no influence on the tested response pattern (Fig. 9B). DC pretreated with LPS either in vitro or in vivo thus down-regulate IFN- release in response to IL-12/IL-18 stimulation.

View larger version (23K): [in this window] [in a new window]   FIGURE 9. In vivo LPS-pretreated splenic DC down-regulate IFN- release in response to IL-12/IL-18 stimulation. A, Splenic CD11c+ DC from B6 mice injected 6 h previously i.v. with 50 µg LPS were isolated as described in Materials and Methods. In flow cytometric analyses, these DC showed up-regulated surface expression of CD40, CD86, and MHC class II molecules. B, Purified splenic DC from either PBS-treated (pretreatment in vivo: none) or LPS-pretreated (pretreatment in vivo: LPS) mice were cultured in vitro for 24 h in GM-CSF (10 ng/ml)-supplemented medium. To the cultures were further added IL-12 (20 ng/ml), IL-18 (60 ng/ml), LPS (1 µg/ml), or CD40L-expressing J558L transfectants, or combinations of these reagents. Supernatants were harvested, and IL-12 p40, IL-12 p70, and IFN- were determined by double-sandwich ELISA. Mean values of triplicates (±SD) of one of three independent experiments are shown.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Within the cultured DC populations, a subset of 20–30% of the cells showed up-regulated MHC class II, CD40, CD80, and CD86 surface expression (Figs. 1 and 2). Furthermore, we detected low but reproducible levels of spontaneous IL-12 and IL-18 (but no IFN-) release in nonstimulated DC cultures. The spontaneously released IL-12 levels were 100- to 300-fold lower than those inducible by CD40 ligation. Despite the use of serum-free medium and rigorous depletion of T cells, B cells, and maturing myeloid cells from the BM progenitor population, evidence for spontaneous DC maturation resulting from endogenous production of maturation factors, triggering through DC interactions, and/or the lack of maturation-suppressing factors was thus apparent in the system confirming previous experience with DC cultures (28). It seems difficult to control spontaneous DC differentiation using currently available DC separation and culture techniques.

IL-12 release by DC is well characterized. Stimulation of DC by GM-CSF and TNF- or LPS (but not GM-CSF and IL-4, IL-18, or IFN) triggers IL-12 release (Fig. 3A and data not shown) (8). Although this pathway leads preferentially to IL-12 p40 release (as described in Ref. 53), we also found some release of IL-12 p70 by LPS-stimulated DC (Fig. 3A). Spontaneous IL-12 release by DC was enhanced 5- to 10-fold by TNF- or LPS stimulation. In contrast, it was enhanced >100-fold by CD40 ligation (Fig. 3A). This confirms that CD40-dependent signals are the most potent stimuli for IL-12 release by DC (23, 24, 25, 26, 27). Although lymphoid-related DC have often been considered the main source of DC-derived IL-12, our data indicate that mDC are also potent producers of IL-12. We describe that BM-derived DC stimulated by CD40 ligation produce a large excess of immunosuppressive IL-12 p40. IL-12 p40 homodimers inhibit Th1-polarized immune responses by suppressing IFN- release from spleen cells, CTL priming, IgG2/IgG3 Ab responses, and allospecific delayed-type hypersensitivity reactions and prolonging allograft survival (53, 54, 55). Because IL-12 p70 secreted by DC stimulated IFN- release (the response was blocked by an anti-IL-12 p35 Ab), its bioactivity was readily detectable in the presence of excess IL-12 p40.

IL-18 is produced by DC (35), but the stimuli that trigger IL-18 expression by DC are not well defined. Spontaneous IL-18 release by DC is detectable but is not up-regulated by stimulation with cytokines, LPS, or CD40 ligation (data not shown). IL-12 produced by mDC in response to CD40 ligation supports their IFN- response. In contrast, endogenous IL-18 release by mDC is insufficient to support IFN- expression by mDC (because exogenous IL-18 had to be provided to detect this response). The release of IL-18 by DC may be low, the secreted IL-18 may be rapidly cleared from the supernatant by DC, and/or the main stimulus that drives IL-18 release by DC may not have been identified. IL-18 is an interesting cytokine: it drives Th2-biased responses of the innate and specific immune system, but strikingly synergizes with IL-12 in driving Th1-polarized immune responses (56). Thus it is a key mediator that can switch a prevailing Th2- into a Th1-biased micromilieu if IL-12 appears.

DC do not release IFN- spontaneously or in response to stimulation by IFN, IL-4, IL-12, IL-18, LPS, or CD40 surface molecules (data not shown). We show in this study the expression and release of IFN- by murine DC stimulated with IL-12 and IL-18. This confirms and extends reports of IFN- production by CD8⁺ lymphoid DC (40) and murine splenic DC populations (41). FACS analyses indicate that 15–20% of the stimulated CD11c⁺ DC population express IFN- (Fig. 4D). We did not detect a marker profile characteristic for IFN--producing DC. In contrast to a previous report (41), IFN- release was readily inducible in CD8⁺ and CD8^- splenic DC (Fig. 8). Hence, IFN- is produced by many cell types stimulated with IL-12 and IL-18, including NK cells, B cells, DC, and macrophages.

The presence of LPS had no effect on the IFN- release of DC stimulated by IL-12/IL-18 or CD40 ligation/IL-18. In contrast, pretreating DC in vitro or in vivo with LPS strikingly down-regulated their ability to respond with IFN- release to stimulation by IL-12/IL-18 or CD40 ligation/IL-18. DC have been shown to produce IL-12 only transiently and to become refractory to further stimulation (57). This exhaustion of cytokine production is also evident from our data and is particularly striking for IFN- production. This observation has implications for T cell priming conditions in, e.g., gut or liver, where continuous LPS exposure prevails.

Our data suggest a cascade of events. Immature mDC spontaneously release low levels of IL-12, IL-18, and chemokines (but no IFN-); it is uncertain whether this takes place also in vivo or whether it reflects in vitro culture conditions that activate DC. Most proinflammatory cytokines tested up-regulate surface expression of MHC and costimulator molecules on the surface of DC but do not stimulate their release of Th1-promoting cytokines (IL-12, IL-18, or IFN-). Ligation of CD40 on the surface of DC is the most potent stimulus for IL-12 p70 release by DC but does not enhance IL-18 or IFN- release. IL-12 p70 confers IL-18 responsiveness to DC by inducing the IL-18 receptor. The synergistic action of IL-12 p70 and IL-18 induces IFN- expression in DC. Thus a cascade of cytokines and cellular interactions is required to induce and/or enhance the Th1-promoting capacity of mDC. These data may help to condition DC to enhance their efficacy in specific, adoptive immunotherapies of cancer, autoimmunity, and chronic infectious disease.

	Acknowledgments

 
The expert technical assistance of Anja Müller is greatly appreciated. We thank Dr. M. J. Grusby (Harvard Medical School) for the BALB/c STAT4^-/- KO mice and Dr. M. J. Wick (Lund, Sweden) for critically reviewing the manuscript and many helpful suggestions.

	Footnotes

 
¹ This work was supported by grants from the Deutsche Forschungsgemeinschaft (Graduiertenkolleg) and the Federal Ministry of Research (DLR 01 GE9907; to J.R. and R.S.).

² Address correspondence and reprint requests to Dr. Jörg Reimann, Department of Medical Microbiology and Immunology, University of Ulm, Helmholtzstrasse 8/1, D-89081 Ulm, Germany. E-mail address: joerg.reimann{at}medizin.uni-ulm.de

³ Abbreviations used in this paper: DC, dendritic cells; mDC, myeloid DC; BM, bone marrow; BMC, BM cells; FL, Flt3 ligand; KO, knockout; CD40L, CD40 ligand.

Received for publication July 6, 2000. Accepted for publication May 1, 2001.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Kalinski, P., C. M. Hilkens, E. Wierenga, M. L. Kapsenberg. 2000. T-cell priming by type-1 and type-2 polarized dendritic cells: the concept of a third signal. Immunol. Today 20:561.
2. Steinman, R. M., M. Pack, K. Inaba. 1997. Dendritic cell development and maturation. Adv. Exp. Med. Biol. 417:1.[Medline]
3. Sallusto, F., A. Lanzavecchia. 1999. Mobilizing dendritic cells for tolerance, priming, and chronic inflammation. J. Exp. Med. 189:611.[Free Full Text]
4. Inaba, K., M. Inaba, N. Romani, H. Aya, M. Deguchi, S. Ikehara, S. Muramatsu, R. M. Steinman. 1992. Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. J. Exp. Med. 176:1693.[Abstract/Free Full Text]
5. Szabolcs, P., M. A. Moore, J. W. Young. 1995. Expansion of immunostimulatory dendritic cells among the myeloid progeny of human CD34⁺ bone marrow precursors cultured with c-kit ligand, granulocyte-macrophage colony-stimulating factor, and TNF-. J. Immunol. 154:5851.[Abstract]
6. Shurin, M. R., P. P. Pandharipande, T. D. Zorina, C. Haluszczak, V. M. Subbotin, O. Hunter, A. Brumfield, W. J. Storkus, E. Maraskovsky, M. T. Lotze. 1997. FLT3 ligand induces the generation of functionally active dendritic cells in mice. Cell. Immunol. 179:174.[Medline]
7. Winzler, C., P. Rovere, M. Rescigno, F. Granucci, G. Penna, L. Adorini, V. S. Zimmermann, J. Davoust, C. P. Ricciardi. 1997. Maturation stages of mouse dendritic cells in growth factor-dependent long-term cultures. J. Exp. Med. 185:317.[Abstract/Free Full Text]
8. Kelleher, P., S. C. Knight. 1998. IL-12 increases CD80 expression and the stimulatory capacity of bone marrow-derived dendritic cells. Int. Immunol. 10:749.[Abstract/Free Full Text]
9. Labeur, M. S., B. Roters, B. Pers, A. Mehling, T. A. Luger, T. Schwarz, S. Grabbe. 1999. Generation of tumor immunity by bone marrow-derived dendritic cells correlates with dendritic cell maturation stage. J. Immunol. 162:168.[Abstract/Free Full Text]
10. Heinzel, F. P., D. S. Schoenhaut, R. M. Rerko, L. E. Rosser, M. K. Gately. 1993. Recombinant interleukin 12 cures mice infected with Leishmania major. J. Exp. Med. 177:1505.[Abstract/Free Full Text]
11. Trinchieri, G.. 1995. Interleukin-12: a proinflammatory cytokine with immunoregulatory functions that bridge innate resistance and antigen-specific adaptive immunity. Annu. Rev. Immunol. 13:251.[Medline]
12. Heufler, C., F. Koch, U. Stanzl, G. Topar, M. Wysocka, G. Trinchieri, A. H. Enk, R. M. Steinman, N. Romani, G. Schuler. 1996. Interleukin-12 is produced by dendritic cells and mediates T helper 1 development as well as interferon- production by T helper 1 cells. Eur. J. Immunol. 26:659.[Medline]
13. Shibuya, K., D. Robinson, F. Zonin, S. B. Hartley, S. E. Macatonia, C. Somoza, C. A. Hunter, K. M. Murphy, A. O’Garra. 1998. IL-1 and TNF are required for IL-12-induced development of Th1 cells producing high levels of IFN- in BALB/c but not C57BL/6 mice. J. Immunol. 160:1708.[Abstract/Free Full Text]
14. Sin, J. I., J. J. Kim, R. L. Arnold, K. E. Shroff, D. McCallus, C. Pachuk, S. P. McElhiney, M. W. Wolf, S. J. Pompa-de Bruin, T. J. Higgins, et al 1999. IL-12 gene as a DNA vaccine adjuvant in a herpes mouse model: IL-12 enhances Th1-type CD4⁺ T cell-mediated protective immunity against herpes simplex virus-2 challenge. J. Immunol. 162:2912.[Abstract/Free Full Text]
15. Gajewski, T. F., J. C. Renauld, A. van Pel, T. Boon. 1995. Costimulation with B7-1, IL-6, and IL-12 is sufficient for primary generation of murine antitumor cytolytic T lymphocytes in vitro. J. Immunol. 154:5637.[Abstract]
16. Bhardwaj, N., R. A. Seder, A. Reddy, M. V. Feldman. 1996. IL-12 in conjunction with dendritic cells enhances antiviral CD8⁺ CTL responses in vitro. J. Clin. Invest. 98:715.[Medline]
17. Eberl, G., B. Kessler, L. P. Eberl, M. J. Brunda, D. Valmori, G. Corradin. 1996. Immunodominance of cytotoxic T lymphocyte epitopes co-injected in vivo and modulation by interleukin-12. Eur. J. Immunol. 26:2709.[Medline]
18. Schmidt, C. S., M. F. Mescher. 1999. Adjuvant effect of IL-12: conversion of peptide antigen administration from tolerizing to immunizing for CD8⁺ T cells in vivo. J. Immunol. 163:2561.[Abstract/Free Full Text]
19. Kaplan, M. H., Y. L. Sun, T. Hoey, M. J. Grusby. 1996. Impaired IL-12 responses and enhanced development of Th2 cells in Stat4-deficient mice. Nature 382:174.[Medline]
20. Magram, J., S. E. Connaughton, R. R. Warrier, D. M. Carvajal, C. Y. Wu, J. Ferrante, C. Stewart, U. Sarmiento, D. A. Faherty, M. K. Gately. 1996. IL-12-deficient mice are defective in IFN production and type 1 cytokine responses. Immunity 4:471.[Medline]
21. Mattner, F., J. Magram, J. Ferrante, P. Launois, K. Di-Padova, R. Behin, M. K. Gately, J. A. Louis, G. Alber. 1996. Genetically resistant mice lacking interleukin-12 are susceptible to infection with Leishmania major and mount a polarized Th2 cell response. Eur. J. Immunol. 26:1553.[Medline]
22. Gately, M. K., L. M. Renzetti, J. Magram, A. S. Stern, L. Adorini, U. Gubler, D. H. Presky. 1998. Interleukin-12/interleukin-12-receptor system: role in normal and pathologic immune responses. Annu. Rev. Immunol. 16:495.[Medline]
23. Armant, M., R. Armitage, N. Boiani, G. Delespesse, M. Sarfati. 1996. Functional CD40 ligand expression on T lymphocytes in the absence of T cell receptor engagement: involvement in interleukin-2-induced interleukin-12 and interferon- production. Eur. J. Immunol. 26:1430.[Medline]
24. Cella, M., D. Scheidegger, L. K. Palmer, 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]
25. DeKruyff, R. H., R. S. Gieni, D. T. Umetsu. 1997. Antigen-driven but not lipopolysaccharide-driven IL-12 production in macrophages requires triggering of CD40. J. Immunol. 158:359.[Abstract]
26. Maruo, S., H. M. Oh, H. J. Ahn, S. Ono, M. Wysocka, Y. Kaneko, H. Yagita, K. Okumura, H. Kikutani, T. Kishimoto, et al 1997. B cells regulate CD40 ligand-induced IL-12 production in antigen-presenting cells (APC) during T cell/APC interactions. J. Immunol. 158:120.[Abstract]
27. Mackey, M. F., J. R. Gunn, C. Maliszewsky, H. Kikutani, R. J. Noelle, J. Barth. 1998. Dendritic cells require maturation via CD40 to generate protective antitumor immunity. J. Immunol. 161:2094.[Abstract/Free Full Text]
28. Schulz, O., D. A. Edwards, M. Schito, J. Aliberti, S. Manickasingham, A. Sher, C. Reis e Sousa. 2000. CD40 triggering of heterodimeric IL-12 p70 production by dendritic cells in vivo requires a microbial priming signal. Immunity 13:453.[Medline]
29. Bradley, L. M., D. K. Dalton, M. Croft. 1996. A direct role for IFN- in regulation of Th1 cell development. J. Immunol. 157:1350.[Abstract]
30. Micallef, M. J., T. Ohtsuki, K. Kohno, F. Tanabe, S. Ushio, M. Namba, T. Tanimoto, K. Torigoe, M. Fujii, M. Ikeda, et al 1996. Interferon--inducing factor enhances T helper 1 cytokine production by stimulated human T cells: synergism with interleukin-12 for interferon- production. Eur. J. Immunol. 26:1647.[Medline]
31. Barbulescu, K., C. Becker, J. F. Schlaak, E. Schmitt, K. H. Meyer-Zum Buschenfelde, M. F. Neurath. 1998. IL-12 and IL-18 differentially regulate the transcriptional activity of the human IFN- promoter in primary CD4⁺ T lymphocytes. J. Immunol. 160:3642.[Abstract/Free Full Text]
32. Coughlin, C. M., K. E. Salhany, M. Wysocka, E. Aruga, H. Kurzawa, A. E. Chang, C. A. Hunter, J. C. Fox, G. Trinchieri, W. F. Lee. 1998. Interleukin-12 and interleukin-18 synergistically induce murine tumor regression which involves inhibition of angiogenesis. J. Clin. Invest. 101:1441.[Medline]
33. Kohno, K., J. Kataoka, T. Ohtsuki, Y. Suemoto, I. Okamoto, M. Usui, M. Ikeda, M. Kurimoto. 1997. IFN-inducing factor (IGIF) is a costimulatory factor on the activation of Th1 but not Th2 cells and exerts its effect independently of IL-12. J. Immunol. 158:1541.[Abstract]
34. Yoshimoto, T., K. Takeda, T. Tanaka, K. Ohkusu, S. Kashiwamura, H. Okamura, S. Akira, K. Nakanishi. 1998. IL-12 up-regulates IL-18 receptor expression on T cells, Th1 cells, and B cells: synergism with IL-18 for IFN- production. J. Immunol. 161:3400.[Abstract/Free Full Text]
35. Stoll, S., H. Jonuleit, E. Schmitt, G. Muller, H. Yamauchi, M. Kurimoto, J. Knop, A. H. Enk. 1998. Production of functional IL-18 by different subtypes of murine and human dendritic cells (DC): DC-derived IL-18 enhances IL-12-dependent Th1 development. Eur. J. Immunol. 28:3231.[Medline]
36. Yoshimoto, T., H. Okamura, Y. I. Tagawa, Y. Iwakura, K. Nakanishi. 1997. Interleukin 18 together with interleukin 12 inhibits IgE production by induction of interferon- production from activated B cells. Proc. Natl. Acad. Sci. USA 94:3948.[Abstract/Free Full Text]
37. Takeda, K., H. Tsutsui, T. Yoshimoto, O. Adachi, N. Yoshida, T. Kishimoto, H. Okamura, K. Nakanishi, S. Akira. 1998. Defective NK cell activity and Th1 response in IL-18-deficient mice. Immunity 8:383.[Medline]
38. Munder, M., M. Mallo, K. Eichmann, M. Modolell. 1998. Murine macrophages secrete interferon- upon combined stimulation with interleukin (IL)-12 and IL-18: a novel pathway of autocrine macrophage activation. J. Exp. Med. 187:2103.[Abstract/Free Full Text]
39. Schindler, H., M. B. Lutz, M. Rollinghoff, C. Bogdan. 2001. The Production of IFN- by IL-12/IL-18-activated macrophages requires STAT4 signaling and is inhibited by IL-4. J. Immunol. 166:3075.[Abstract/Free Full Text]
40. Ohteki, T., T. Fukao, K. Suzue, C. Maki, M. Ito, M. Nakamura, S. Koyasu. 1999. Interleukin 12-dependent interferon- production by CD8⁺ lymphoid dendritic cells. J. Exp. Med. 189:1981.[Abstract/Free Full Text]
41. Fukao, T., S. Matsuda, S. Koyasu. 2000. Synergistic effects of IL-4 and IL-18 on IL-12-dependent IFN- production by dendritic cells. J. Immunol. 164:64.[Abstract/Free Full Text]
42. Maraskovsky, E., K. Brasel, M. Teepe, E. R. Roux, S. D. Lyman, K. Shortman, H. J. McKenna. 1996. Dramatic increase in the numbers of functionally mature dendritic cells in Flt3 ligand-treated mice: multiple dendritic cell subpopulations identified. J. Exp. Med. 184:1953.[Abstract/Free Full Text]
43. Pulendran, B., J. Lingappa, M. K. Kennedy, J. Smith, M. Teepe, A. Rudensky, C. R. Maliszewski, E. Maraskovsky. 1997. Developmental pathways of dendritic cells in vivo: distinct function, phenotype, and localization of dendritic cell subsets in FLT3 ligand-treated mice. J. Immunol. 159:2222.[Abstract/Free Full Text]
44. Vremec, D., K. Shortman. 1997. Dendritic cell subtypes in mouse lymphoid organs: cross-correlation of surface markers, changes with incubation, and differences among thymus, spleen, and lymph nodes. J. Immunol. 159:565.[Abstract]
45. Caux, C., D. C. Dezutter, D. Schmitt, J. Banchereau. 1992. GM-CSF and TNF- cooperate in the generation of dendritic Langerhans cells. Nature 360:258.[Medline]
46. Caux, C., B. Vanbervliet, C. Massacrier, D. C. Dezutter, B. de Saint-Vis, C. Jacquet, K. Yoneda, S. Imamura, D. Schmitt, J. Banchereau. 1996. CD34⁺ hematopoietic progenitors from human cord blood differentiate along two independent dendritic cell pathways in response to GM-CSF+TNF. J. Exp. Med. 184:695.[Abstract/Free Full Text]
47. Jonuleit, H., U. Kuhn, G. Muller, K. Steinbrink, L. Paragnik, E. Schmitt, J. Knop, A. H. Enk. 1997. Pro-inflammatory cytokines and prostaglandins induce maturation of potent immunostimulatory dendritic cells under fetal calf serum-free conditions. Eur. J. Immunol. 27:3135.[Medline]
48. Geissmann, F., C. Prost, J. P. Monnet, M. Dy, N. Brousse, O. Hermine. 1998. Transforming growth factor 1, in the presence of granulocyte/macrophage colony-stimulating factor and interleukin 4, induces differentiation of human peripheral blood monocytes into dendritic Langerhans cells. J. Exp. Med. 187:961.[Abstract/Free Full Text]
49. Delneste, Y., N. Herbault, B. Galea, G. Magistrelli, I. Bazin, J. Y. Bonnefoy, P. Jeannin. 1999. Vasoactive intestinal peptide synergizes with TNF- in inducing human dendritic cell maturation. J. Immunol. 163:3071.[Abstract/Free Full Text]
50. Seguin, R., L. H. Kasper. 1999. Sensitized lymphocytes and CD40 ligation augment interleukin-12 production by human dendritic cells in response to Toxoplasma gondii. J. Infect. Dis. 179:467.[Medline]
51. Vremec, D., J. Pooley, H. Hochrein, L. Wu, K. Shortman. 2000. CD4 and CD8 expression by dendritic cell subtypes in mouse thymus and spleen. J. Immunol. 164:2978.[Abstract/Free Full Text]
52. Inaba, K., M. Inaba, M. Deguchi, K. Hagi, R. Yasumizu, S. Ikehara, S. Muramatsu, R. M. Steinman. 1993. Granulocytes, macrophages, and dendritic cells arise from a common major histocompatibility complex class II-negative progenitor in mouse bone marrow. Proc. Natl. Acad. Sci. USA 90:3038.[Abstract/Free Full Text]
53. Heinzel, F. P., A. M. Hujer, F. N. Ahmed, R. M. Rerko. 1997. In vivo production and function of IL-12 p40 homodimers. J. Immunol. 158:4381.[Abstract]
54. Kato, K., O. Shimozato, K. Hoshi, H. Wakimoto, H. Hamada, H. Yagita, K. Okumura. 1996. Local production of the p40 subunit of interleukin 12 suppresses T-helper 1-mediated immune responses and prevents allogeneic myoblast rejection. Proc. Natl. Acad. Sci. USA 93:9085.[Abstract/Free Full Text]
55. Abdi, K., S. H. Herrmann. 1997. CTL generation in the presence of IL-4 is inhibited by free p40: evidence for early and late IL-12 function. J. Immunol. 159:3148.[Abstract]
56. Nakanishi, K.. 2001. Innate and acquired activation pathways in T cells. Nat. Immunol. 2:140.[Medline]
57. Langenkamp, A., M. Messi, A. Lanzavecchia, F. Sallusto. 2000. Kinetics of dendritic cell activation: impact on priming of Th1, Th2 and nonpolarized T cells. Nat. Immunol. 1:311.[Medline]

This article has been cited by other articles:

				J. Barbi, H. M. Snider, N. Bhardwaj, C. M. Lezama-Davila, J. E. Durbin, and A. R. Satoskar Signal transducer and activator of transcription 1 in T cells plays an indispensable role in immunity to Leishmania major by mediating Th1 cell homing to the site of infection FASEB J, November 1, 2009; 23(11): 3990 - 3999. [Abstract] [Full Text] [PDF]

				E. A. Haddad, L. K. Senger, and F. Takei An Accessory Role for B Cells in the IL-12-Induced Activation of Resting Mouse NK Cells J. Immunol., September 15, 2009; 183(6): 3608 - 3615. [Abstract] [Full Text] [PDF]

				A. G. Joyee, H. Qiu, Y. Fan, S. Wang, and X. Yang Natural Killer T Cells Are Critical for Dendritic Cells to Induce Immunity in Chlamydial Pneumonia Am. J. Respir. Crit. Care Med., October 1, 2008; 178(7): 745 - 756. [Abstract] [Full Text] [PDF]

				K. Suzue, S. Kobayashi, T. Takeuchi, M. Suzuki, and S. Koyasu Critical role of dendritic cells in determining the Th1/Th2 balance upon Leishmania major infection Int. Immunol., March 1, 2008; 20(3): 337 - 343. [Abstract] [Full Text] [PDF]

				C. Antonopoulos, M. Cumberbatch, J. B. Mee, R. J. Dearman, X.-Q. Wei, F. Y. Liew, I. Kimber, and R. W. Groves IL-18 is a key proximal mediator of contact hypersensitivity and allergen-induced Langerhans cell migration in murine epidermis J. Leukoc. Biol., February 1, 2008; 83(2): 361 - 367. [Abstract] [Full Text] [PDF]

				M. C. Siracusa, M. G. Overstreet, F. Housseau, A. L. Scott, and S. L. Klein 17{beta}-Estradiol Alters the Activity of Conventional and IFN-Producing Killer Dendritic Cells J. Immunol., February 1, 2008; 180(3): 1423 - 1431. [Abstract] [Full Text] [PDF]

				A. Maroof and P. M. Kaye Temporal Regulation of Interleukin-12p70 (IL-12p70) and IL-12-Related Cytokines in Splenic Dendritic Cell Subsets during Leishmania donovani Infection Infect. Immun., January 1, 2008; 76(1): 239 - 249. [Abstract] [Full Text] [PDF]

				A. L. Coelho, M. A. Schaller, C. F. Benjamim, A. Z. Orlofsky, C. M. Hogaboam, and S. L. Kunkel The Chemokine CCL6 Promotes Innate Immunity via Immune Cell Activation and Recruitment J. Immunol., October 15, 2007; 179(8): 5474 - 5482. [Abstract] [Full Text] [PDF]

				M. M. Moretto, L. M. Weiss, C. L. Combe, and I. A. Khan IFN-{gamma}-Producing Dendritic Cells Are Important for Priming of Gut Intraepithelial Lymphocyte Response Against Intracellular Parasitic Infection J. Immunol., August 15, 2007; 179(4): 2485 - 2492. [Abstract] [Full Text] [PDF]

				L. Li, L. Huang, S.-s. J. Sung, P. I. Lobo, M. G. Brown, R. K. Gregg, V. H. Engelhard, and M. D. Okusa NKT Cell Activation Mediates Neutrophil IFN-{gamma} Production and Renal Ischemia-Reperfusion Injury J. Immunol., May 1, 2007; 178(9): 5899 - 5911. [Abstract] [Full Text] [PDF]

				S.-R. Chang, K.-J. Wang, Y.-F. Lu, L.-J. Yang, W.-J. Chen, Y.-H. Lin, H.-H. Chang, and S.-L. Wang Characterization of Early Gamma Interferon (IFN-{gamma}) Expression during Murine Listeriosis: Identification of NK1.1+ CD11c+ Cells as the Primary IFN-{gamma}-Expressing Cells Infect. Immun., March 1, 2007; 75(3): 1167 - 1176. [Abstract] [Full Text] [PDF]

				D. Vremec, M. O'Keeffe, H. Hochrein, M. Fuchsberger, I. Caminschi, M. Lahoud, and K. Shortman Production of interferons by dendritic cells, plasmacytoid cells, natural killer cells, and interferon-producing killer dendritic cells Blood, February 1, 2007; 109(3): 1165 - 1173. [Abstract] [Full Text] [PDF]

				U. I. Chaudhry, T. P. Kingham, G. Plitas, S. C. Katz, J. R. Raab, and R. P. DeMatteo Combined Stimulation with Interleukin-18 and CpG Induces Murine Natural Killer Dendritic Cells to Produce IFN-{gamma} and Inhibit Tumor Growth Cancer Res., November 1, 2006; 66(21): 10497 - 10504. [Abstract] [Full Text] [PDF]

				H. Li, W. Wojciechowski, C. Dell'Agnola, N. E. Lopez, and I. Espinoza-Delgado IFN-{gamma} and T-bet Expression in Human Dendritic Cells from Normal Donors and Cancer Patients Is Controlled through Mechanisms Involving ERK-1/2-Dependent and IL-12-Independent Pathways J. Immunol., September 15, 2006; 177(6): 3554 - 3563. [Abstract] [Full Text] [PDF]

				S. E. Moreno, J. C. Alves-Filho, T. M. Alfaya, J. S. da Silva, S. H. Ferreira, and F. Y. Liew IL-12, but Not IL-18, Is Critical to Neutrophil Activation and Resistance to Polymicrobial Sepsis Induced by Cecal Ligation and Puncture. J. Immunol., September 1, 2006; 177(5): 3218 - 3224. [Abstract] [Full Text] [PDF]

				J. R. Maxwell, R. Yadav, R. J. Rossi, C. E. Ruby, A. D. Weinberg, H. L. Aguila, and A. T. Vella IL-18 Bridges Innate and Adaptive Immunity through IFN-{gamma} and the CD134 Pathway J. Immunol., July 1, 2006; 177(1): 234 - 245. [Abstract] [Full Text] [PDF]

				U. I. Chaudhry, S. C. Katz, T. P. Kingham, V. G. Pillarisetty, J. R. Raab, A. B. Shah, and R. P. DeMatteo In vivo overexpression of Flt3 ligand expands and activates murine spleen natural killer dendritic cells FASEB J, May 1, 2006; 20(7): 982 - 984. [Abstract] [Full Text] [PDF]

				I. Fricke, D. Mitchell, J. Mittelstadt, N. Lehan, H. Heine, T. Goldmann, A. Bohle, and S. Brandau Mycobacteria Induce IFN-{gamma} Production in Human Dendritic Cells via Triggering of TLR2 J. Immunol., May 1, 2006; 176(9): 5173 - 5182. [Abstract] [Full Text] [PDF]

				R. E. Berg, E. Crossley, S. Murray, and J. Forman Relative Contributions of NK and CD8 T Cells to IFN-{gamma} Mediated Innate Immune Protection against Listeria monocytogenes J. Immunol., August 1, 2005; 175(3): 1751 - 1757. [Abstract] [Full Text] [PDF]

				C. Tenger, A. Sundborger, J. Jawien, and X. Zhou IL-18 Accelerates Atherosclerosis Accompanied by Elevation of IFN-{gamma} and CXCL16 Expression Independently of T Cells Arterioscler Thromb Vasc Biol, April 1, 2005; 25(4): 791 - 796. [Abstract] [Full Text] [PDF]

				V. G. Pillarisetty, S. C. Katz, J. I. Bleier, A. B. Shah, and R. P. DeMatteo Natural Killer Dendritic Cells Have Both Antigen Presenting and Lytic Function and in Response to CpG Produce IFN-{gamma} via Autocrine IL-12 J. Immunol., March 1, 2005; 174(5): 2612 - 2618. [Abstract] [Full Text] [PDF]

				U. Schleicher, A. Hesse, and C. Bogdan Minute numbers of contaminant CD8+ T cells or CD11b+CD11c+ NK cells are the source of IFN-{gamma} in IL-12/IL-18-stimulated mouse macrophage populations Blood, February 1, 2005; 105(3): 1319 - 1328. [Abstract] [Full Text] [PDF]

				N. M. Tsuji, H. Tsutsui, E. Seki, K. Kuida, H. Okamura, K. Nakanishi, and R. A. Flavell Roles of caspase-1 in Listeria infection in mice Int. Immunol., February 1, 2004; 16(2): 335 - 343. [Abstract] [Full Text] [PDF]

				P.-H. Chiang, L. Wang, C. A. Bonham, X. Liang, J. J. Fung, L. Lu, and S. Qian Mechanistic Insights into Impaired Dendritic Cell Function by Rapamycin: Inhibition of Jak2/Stat4 Signaling Pathway J. Immunol., February 1, 2004; 172(3): 1355 - 1363. [Abstract] [Full Text] [PDF]

				B.-G. Xiao, X.-C. Wu, J.-S. Yang, L.-Y. Xu, X. Liu, Y.-M. Huang, B. Bjelke, and H. Link Therapeutic potential of IFN-{gamma}-modified dendritic cells in acute and chronic experimental allergic encephalomyelitis Int. Immunol., January 1, 2004; 16(1): 13 - 22. [Abstract] [Full Text] [PDF]

				Z. Guo, M. Zhang, H. An, W. Chen, S. Liu, J. Guo, Y. Yu, and X. Cao Fas ligation induces IL-1{beta}-dependent maturation and IL-1{beta}-independent survival of dendritic cells: different roles of ERK and NF-{kappa}B signaling pathways Blood, December 15, 2003; 102(13): 4441 - 4447. [Abstract] [Full Text] [PDF]

				R. E. Berg, E. Crossley, S. Murray, and J. Forman Memory CD8+ T Cells Provide Innate Immune Protection against Listeria monocytogenes in the Absence of Cognate Antigen J. Exp. Med., November 17, 2003; 198(10): 1583 - 1593. [Abstract] [Full Text] [PDF]

				G. Lugo-Villarino, R. Maldonado-Lopez, R. Possemato, C. Penaranda, and L. H. Glimcher T-bet is required for optimal production of IFN-{gamma} and antigen-specific T cell activation by dendritic cells PNAS, June 24, 2003; 100(13): 7749 - 7754. [Abstract] [Full Text] [PDF]

				T. K. Means, F. Hayashi, K. D. Smith, A. Aderem, and A. D. Luster The Toll-Like Receptor 5 Stimulus Bacterial Flagellin Induces Maturation and Chemokine Production in Human Dendritic Cells J. Immunol., May 15, 2003; 170(10): 5165 - 5175. [Abstract] [Full Text] [PDF]

				D. Stober, I. Jomantaite, R. Schirmbeck, and J. Reimann NKT Cells Provide Help for Dendritic Cell-Dependent Priming of MHC Class I-Restricted CD8+ T Cells In Vivo J. Immunol., March 1, 2003; 170(5): 2540 - 2548. [Abstract] [Full Text] [PDF]

				J. M. Lee, A. Mahtabifard, R. Yamada, R. G. Crystal, and R. J. Korst Adenovirus Vector-mediated Overexpression of a Truncated Form of the p65 Nuclear Factor {kappa}B cDNA in Dendritic Cells Enhances Their Function Resulting in Immune-mediated Suppression of Preexisting Murine Tumors Clin. Cancer Res., November 1, 2002; 8(11): 3561 - 3569. [Abstract] [Full Text] [PDF]

				P. Riedl, D. Stober, C. Oehninger, K. Melber, J. Reimann, and R. Schirmbeck Priming Th1 Immunity to Viral Core Particles Is Facilitated by Trace Amounts of RNA Bound to Its Arginine-Rich Domain J. Immunol., May 15, 2002; 168(10): 4951 - 4959. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Stober, D. Articles by Reimann, J. Search for Related Content PubMed PubMed Citation Articles by Stober, D. Articles by Reimann, J.