Dendritic cells (DCs) are major antigen-presenting cells of the immune system, which need to be activated in order to initiate an immune response. Here, we describe the immunostimulatory effects on human monocyte-derived DCs observed upon infection with Listeria monocytogenes or after treatment with listerial lipoteichoic acid (LTA) and lipopolysaccharide (LPS), respectively. All stimuli caused upregulation of costimulatory molecules, induced T-cell proliferative responses and secretion of cytokines in vitro. Infection of DCs with L. monocytogenes induced release of interleukin (IL)-12 and IL-18. In contrast treatment with purified listerial LTA yielded high levels of IL-18 release, but only minimal IL-12 production. Treatment of DCs with LPS conversely induced significant amounts of IL-12 production, but no IL-18. The release of both stimulating cytokines IL-12 and IL-18 upon infection with entire bacteria suggests that attenuated strains of L. monocytogenes may be a valuable tool for subunit vaccine delivery.
Dendritic cells (DCs) are professional antigen-presenting cells which play a crucial role in initiating an immune response . DCs exist in different functional stages : (1) Immature DCs are characterized by their ability to efficiently take up antigen while their T cell stimulatory function is poor. Placed primarily in tissues such as skin and mucosa, immature DCs are among the first cells to encounter a pathogen, which they will internalize and process before migration into lymphoid organs and maturation of DCs takes place. (2) Mature DCs are potent stimulators of naive T-cells and play a role in their instruction to become Th1 or Th2 cells. Many factors are important for the determination of differentiation into Th1 and Th2 cells. In addition to the type and dose of antigen and the genetic background of the host, the most critical factors remain the type of DCs involved in the interaction with naive T cells, the type of costimulatory molecules expressed on the surface of the DCs, and the polarizing cytokine microenvironment during antigen presentation [1–3].
Maturation of DCs can be induced by various stimuli in vitro. A common procedure is an exposure of immature DCs to a cytokine cocktail containing interleukin (IL)-6, IL-1β, TNF-α, and prostaglandin E2[4–5]. Maturation of DCs can also be induced by infection with various bacteria [6–7]. We recently showed, that infection of DCs with L. monocytogenes causes efficient maturation. Moreover, listerial lipoteichoic acid (LTA) is also capable of inducing maturation of DCs which results in morphologically and phenotypically similar DCs compared to treatment with the cytokine cocktail or infection with L. monocytogenes. LTAs are amphipathic polymers, which consist of alternating glycerol-phosphate units of variable chain length with sugar, amino acid, and fatty acid substitutions. They are retained in the outside of the cytoplasmic membrane by their lipid moiety [9–11].
L. monocytogenes is a Gram-positive, facultative intracellular bacterium and serves as a promising vector for presenting tumor or infection-specific antigens to the immune system. Attenuated strains of L. monocytogenes have been successfully utilized for the delivery of protein antigens and DNA vaccine vectors into the cytosol of antigen presenting cells [12–14].
In this study we show that, despite phenotypic and morphological identities, DCs matured with various stimuli differ in their functional capacities, in particular concerning the secretion of the cytokines IL-12 and IL-18.
2Materials and methods
2.1Isolation of human DCs from peripheral blood
Peripheral blood mononuclear cells (PBMC) were isolated from heparinized leukocyte-enriched buffy coats of healthy adult donors by Lymphoprep (1.077 g ml−1; Nycomed, Oslo, Norway) density gradient centrifugation applying 400×g at room temperature. PBMCs were plated on tissue culture dishes (3003; Falcon Labware, Oxnard, CA, USA) at a density of 5×106 cells ml−1 in RPMI 1640 medium (Gibco/Life Technologies, Karlsruhe, Germany), supplemented with l-glutamine (2 mM), 1% autologous human plasma, and 100 U ml−1 (9 ng ml−1) rhGM-CSF (Leucomax®, Essex-Pharma, München, Germany) for 45 min at 37°C. Nonadherent cells were washed free with warm phosphate-buffered saline, and adherent cells were cultured for 7 days without antibiotics in RPMI 1640 medium, supplemented with 1% autologous human plasma, 2mM l-glutamine, 1000 U ml−1 (10 ng ml−1) of recombinant human (rh) IL-4 (PBH, Hannover, Germany) and 800 U ml−1 (72 ng ml−1) rhGM-CSF. Cytokines were replenished every other day.
2.2Maturation of immature DCs
Maturation of immature DCs was induced in four different ways by adding (1) a cytokine cocktail containing IL-6 (1000 U ml−1 resp. 5 ng ml−1; PBH, Hannover, Germany), IL-1β (1000 U ml−1 resp. 10 ng ml−1; PBH), TNF-α (1000 U ml−1 resp. 10 ng ml−1; PBH), and prostaglandin E2 (PGE2, 10−8 M resp. 1 μg ml−1, Sigma Aldrich Chemie, Steinheim, Germany), (2) listerial LTA fraction I and II (10–100 μg ml−1), and (3) lipopolysaccharide (LPS) (1–10μg, from Escherichia coli Serotype 0128:B12, Sigma-Aldrich Chemie) to the immature DCs cultures for 48 h. Maturation of immature DCs was also induced by infection (4) of cells with wild-type L. monocytogenes Sv 1/2a EGD.
2.3In vitro bacterial infection of DCs
L. monocytogenes EGD wild-type strain, used in this study, was grown in brain heart infusion medium at 37°C with rigorous shaking until they reached the mid-exponential phase of growth. After washing twice with phosphate-buffered saline medium, the bacteria pellets were diluted in RPMI 1640 medium (Gibco/Life Technologies) and were added to immature DCs at a multiplicity of infection (MOI) of 10. The cultures were incubated in RPMI 1640 medium supplemented with 1% autologous human plasma at 37°C for 1 h. For removal of extracellular bacteria, cells were washed twice and cultured in the presence of 50 μg ml−1 gentamicin (Gibco/Life Technologies). Infected DCs were incubated for 48 h.
The cell surface of Listeria strains are composed of various compounds. These include peptidoglycan, teichoic acids and LTA. LTA can be phenol-extracted and purified from Listeria. Hydrophobic chromatography on octyl-Sepharose yielded, in case of L. monocytogenes Sv4b, two fractions, LTA I and LTA II. The latter was found to contain a glycolipid anchor substituted with a phosphatidyl residue that is absent in LTA I [10–11]. Both fractions of listerial LTA used in the experiments were kindly provided by Prof. em. F. Fiedler (Institute for Genetics and Microbiology, Munich, Germany) [9–11].
Briefly, LTA from heat-inactivated and mechanically disintegrated L. monocytogenes (Sv4b) cells were extracted with 40% (v/v) phenol at 65°C for 45 min and the phases separated by centrifugation. The aqueous phase containing LTA was dialyzed against 0.05 M sodium acetate (pH 4.0). LTA was then purified by separation on an octyl-Sepharose CL-4b column (Pharmacia) with a linear gradient [0–66% (v/v) propanol in 0.05 M sodium acetate, pH 4.0]. Phosphate-containing fractions eluted within the propanol gradient were pooled, dialyzed against double distilled water, and lyophilized .
Flow cytometry was used to monitor the expression of surface markers of uninfected, infected, cocktail-stimulated and LTA- or LPS-treated DCs. Indirect immunofluorescence was performed using murine monoclonal antibodies revealed by phycoerythrin- or FITC-conjugated anti-mouse immunoglobulin (Dianova, Hamburg, Germany). The primary antibodies used were α-HLA class II DR/DQ (9.3F10, Manassas, VA, USA), CD83 (HB15a, Pharmingen, Hamburg, Germany), CD86 (IT2.2, Pharmingen). The stained cells were analyzed on an EPICS XL-MCL (Coulter Immunotech Diagnotics, Krefeld, Germany).
2.6Cytokine assessment by ELISA of DCs supernatants
To assess the amounts of cytokines secreted by mature DCs generated with different stimuli, supernatants were collected 48 h after incubation with the stimuli. The secretion of TNF-α, IL-12 (p70) and IL-18 was determined by ELISA according to the manufacturers’ instructions (Bender MedSystems, Vienna, Austria).
2.7RNA purification of total and reverse transcription
Total RNA was prepared using the Qiagen RNeasy Kit (Qiagen GmbH, Hilden, Germany). Purified RNA was eluted with 33 μl DEPC-treated water. For reverse transcription, 3 μl oligo (dT) (Pharmacia Biotech, Freiburg, Germany) were added, incubated for 10 min at 70°C and chilled on ice. After mixing with 12 μl First Strand Buffer, 6 μl 0.1M DTT (Life Technologies), 10 mM dNTPs (Pharmacia Biotech) and 3 μl Superscript II reverse transcriptase (200U μl−1, Life Technologies), the specimens were incubated for 50 min at 42°C. Reverse transcriptase was denaturated by incubation for 10 min at 95°C.
2.8Quantification of β-actin, IL-12, TNF-α, and IL-18 mRNA expression
All PCR reactions were performed on an ABI Prism Sequence Detection System (Perkin Elmer, Foster City, CA, USA). Composition of the PCR assay and oligonucleotide sequences have been published previously .
2.9Mixed lymphocyte reaction (MLR)
All MLRs were performed in round-bottomed microtiter wells in a final volume of 200 μl of RPMI 1640 medium supplemented with l-glutamine (2 mM) and 10% fetal calf serum. In these experiments, 105 cryopreserved allogeneic T-cells, isolated by rosetting with sheep erythrocytes, were mixed with varying numbers of mature DCs. All cells were cultured continuously for 6 days at 37°C. To measure cellular proliferation, 1 μCi [3H]thymidine per well were added to the assays for the last 16 h of incubation. Cells were collected on glassfiber filters by using a Betaplate 96 well harvester (Pharmacia-LKB, Uppsala, Sweden) and incorporated radioactivity was quantified with a 96-well Betaplate liquid scintillation counter (Pharmacia-LKB). Results are expressed as mean counts per minute.
The data are presented as mean values and standard deviations of a representative experiment. All experiments were performed at least three times with cells from different donors.
For statistical comparison, the Student's t test was performed. P values of <0.01 were considered statistically significant.
3Results and discussion
Immature DCs were generated from human PBMC. Quality and purity of the DCs was monitored by flow cytometry. According to the forward scatter-side scatter of the flow cytometry, 80–90% of the cells were CD1a+, HLA class II+, and CD83 negative. These data indicate that the cell population exhibited the typical surface marker pattern of immature DCs .
Maturation of DCs by infection with L. monocytogenes and addition of the described maturating cytokine cocktail, respectively, resulted in an upregulation of CD83, CD86, and MHC II, as previously described . In addition both fractions of listerial LTA (I and II; Fig. 1) as well as LPS induced dose-dependent upregulation of these surface markers.
To assess functional differences of these phenotypically identical mature DCs, we compared the allostimulatory activity of L. monocytogenes-infected, LPS-/LTA-treated, and cytokine cocktail stimulated DCs. As shown in Fig. 2, we could not detect any significant differences in the capacity to stimulate naive T-cells comparing DCs matured by different stimuli. In general, maturation is accompanied with an increase in the allostimulatory potential of the DCs, which was dose-dependent for treatment with listerial LTA fractions.
Next, we investigated the release of the cytokines IL-12, IL-18, and TNF-α, by human monocyte-derived DCs in response to infection with L. monocytogenes, treatment with listerial LTA, treatment with LPS of E. coli, and treatment with a cytokine cocktail. DCs are important immune modulators, which secrete selective cytokines that facilitate acute primary inflammatory responses and antigen presentation. Cytokines released by activated DCs can direct the adaptive T cell response towards either Th1 or Th2 pattern .
Our data show that unstimulated DCs generally produce low levels of TNF-α (50 pg ml−1), IL-12 (p70) (8 pg ml−1) and IL-18 (50 pg ml−1). These cytokine levels were not significantly increased in cytokine-matured DCs. In contrast, infection of human DCs with L. monocytogenes (MOI of 10) in the presence of autologous human plasma (1%) effectively stimulated the release of TNF-α, IL-12 and IL-18 (Fig. 3). These results were confirmed by quantifying the cytokine mRNA expression with RT-PCR: 12 h after infection of DCs with L. monocytogenes mRNA coding for IL-12 (p40) could be detected at relevant amounts (120,000 molecules/1,000,000 molecules β-actin). Immature, uninfected DCs (5 molecules/1,000,000 molecules β-actin), cocktail-stimulated (130 molecules/1,000,000 molecules β-actin) and LTA-treated DCs (50 molecules/1,000,000 molecules β-actin) showed only low expression levels of IL12 (p40) mRNA.
Although no phenotypic differences between Listeria-infected and listerial LTA-treated DCs can be observed, we noticed some remarkable functional differences. The two fractions of listerial LTA differ in their ability to trigger cytokine release. Whereas listerial LTA II (100 μg ml−1) had no effect on IL-12 production, LTA I (100 μg ml−1) induced a slight release of IL-12 (p70). However, the amount of IL-12 induced by LTA I treatment of DCs was significantly lower than that following infection with the entire bacteria.
With respect to IL-18 and TNF-α the addition of LTA I to DCs strongly enhanced the production of both cytokines in a dose-dependent manner. At a concentration of 100 μg ml−1 the amount of IL-18 was twice as high as that obtained by L. monocytogenes-infected DCs. In contrast to LTA I, LTA II (in the range of 10–100 μg ml−1) failed to provoke cytokine release by monocyte derived DCs. The two forms of listerial LTA obtained after octyl-Sepharose chromatography differ in their fatty acid substitutions, with fraction I having two fatty acids and fraction II four fatty acids linked to one end of the glycerol-phosphate backbone [10,18]. It is therefore likely that fatty acid substitutions may influence the binding of LTA to cell receptors [11,19]. The lipid moiety of lipopeptides was found to be essential for induction of DC-maturation .
LPS (1–10 μg ml−1) induced a third pattern of cytokine secretion in DC by provoking a marked stimulation of TNF-α and IL-12 but no IL-18 secretion (Fig. 3).
These differences suggest an alternate pathway of signal transduction in monocyte-derived DCs by LTA and LPS. Receptors have evolved on DCs to recognize conserved molecules on bacteria and other evolutionarily distant organism. A variety of microbial ligands have been shown to activate immune responses via mammalian Toll-like receptors (TLRs) . Monocyte-derived DCs express TLR 1, 2, 4, 5, 8 . Several groups have shown that preparations of Gram-positive bacteria mediate cellular signaling via TLR2. It has been shown that TLR2 can restore the responsiveness of cells to peptidoglycans and LTA . TLR 4 is instrumental for the recognition of LPS [21–23]. It appears that TLRs offer DCs a means of discriminating between different stimuli. However, it is unknown whether signalling through different TLRs leads to different types of adaptive immune responses.
In contrast to LPS, at least 1000-fold higher concentrations of listerial LTA were required for DC maturation. Significant stimulatory effects of LPS are already observed at a concentration lower than 1 ng ml−1. LTAs are inserted into the bacterial cytoplasmic membrane via their fatty acids with the glycerol-phosphate backbone passing through the murein sacculus, probably resulting in a highly ordered complex . The loss of this structure, when adding LTA in solution to DCs, might be one explanation for the high concentration of LTA needed for cytokine activation.
In conclusion, infection of DCs with L. monocytogenes resulted in a significantly higher IL-12 and IL-18 production compared to DCs matured with the standard cytokine cocktail. The cytokines produced by DCs are key in determining the type of T-cell response generated. IL-12 is a major Th1-promoting factor [26–28]. It is essential for the generation of Th1-specific cells from their naive precursors. In addition, IL-12 induces interferon-γ (IFN-γ) production by T cells. Both, IL-12 and IFN-γ are required for the generation of protective Th1-immune response against intracellular bacteria, such as L. monocytogenes[29–30]. IL-18, an IFN-γ inducing factor, plays an important role in cancer inhibition and combating infectious diseases . In combination with IL-12, IL-18 effectively induces IFN-γ and stimulates a Th1-mediated immune response , which is critical in the host defense against infection with intracellular microbes. A critical role for IL-18 has recently been described in L. monocytogenes-infected mice . Infection was greatly exacerbated by anti-IL-18-receptor monoclonal antibody. Moreover, IL-18 was required for the macrophage's subsequent release of nitric oxide in response to L. monocytogenes infection. IL-18-dependent NO production may be a major mechanism of bacterial clearance.
IL-18 is synthesized as biologically inactive precursor. This precursor is cleaved by caspase-1 to form the biologically active mature cytokine . One may speculate that listerial LTA compared to LPS can more efficiently enhance the activation caspase-1.
Recently, L. monocytogenes has been used for the delivery of subunit vaccines. Attenuated L. monocytogenes have been exploited for the delivery of heterologous antigens expressed by the bacterium as well as for the direct delivery of eukaryotic DNA vaccine vectors leading to antigen expression by infected cells [13–14].
This study underlines the potential of L. monocytogenes as a carrier for vaccination vectors. The production of IL-12 and IL-18 by L. monocytogenes-infected DCs provides important adjuvant effects for vaccination purposes aiming at Th-1-type immune responses. This might be of relevance for designing therapeutic strategies, especially with respect to subunit vaccine delivery in this promising cell type for antitumor therapy.
This study was supported by a fellowship from the ‘Bayerisches Staatsministerium für Wissenschaft, Forschung und Kunst’ to A.K.-M. Part of this study was funded by grants of the German Ministry for Education and Research (BMBF 01KX9820/L, IZKF-01KS9603 and SFB465) to E.K. We thank Prof. G. Szalay for critical reading of the manuscript and Dr. N. Kruse for helping with the ABI Prism Sequence Detection System. We are grateful to Prof. F. Fiedler, Munich, for donating the listerial LTA.