1 Institute of Immunology, College of Veterinary Medicine and Biotechnological-Biomedical Center (BBZ), University of Leipzig, An den Tierkliniken 11, 04103 Leipzig, Germany
2 Max Planck Institute of Immunobiology, Research Group for Medical Microbiology, Stübeweg 51, 79011 Freiburg, Germany
Correspondence to: R. K. Straubinger; E-mail: straubinger{at}uni-leipzig.de
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Abstract |
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Keywords: bacteria, cytokine, LPS, mouse, pattern recognition receptor
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Introduction |
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IL-12p70 is essential for induction of the Th1 immune response (5, 6), that is important to protect against intracellular pathogens (710). Experimentally, IL-12-deficient mice show a dramatically impaired IFN- release and a marginal delayed-type hypersensitivity response, demonstrating the importance of this factor for the protection against intracellular bacterial infections (11). The gene loci and promoters of both subunits are quite different, implying a differential regulation of these genes (12, 13) and even functional differences can be observed between the p35- and p40-deficient mice. IL-12p35-deficient mice, however, still develop a marginal Th1 response to Mycobacteria spp. or Salmonella spp. infections. Additional p40-dependent cytokines that facilitate cellular immune responses are in place that help to protect against intracellular microbes (1416).
The search for new members of the IL-12 cytokine family led to the discovery of IL-23 that contains the p40 subunit of IL-12p70 and the newly discovered p19 subunit (17). Transgenic mice for p19 show a multi-organ inflammation, neutrophilia and elevated tumor necrosis factor- (TNF-
) and IL-1 serum levels compared with the non-transgenic littermates (18). It was demonstrated that IL-23, but not IL-12p70, is the critical factor for autoimmune inflammatory responses such as experimental autoimmune encephalomyelitis (EAE) in the brain, characterized by a substantial influx of T cells and CD11b+ monocytes into the central nervous system (19). Furthermore, it was shown that the specific receptor chain for IL-23, the IL-23R, is induced in murine bone marrow-derived macrophages (BMDM
) after exposure to LPS (20), suggesting an as yet unknown response of myeloid cells to IL-23, which supposedly are the early initiators of a pro-inflammatory immune response. On the other hand, IL-23 enhances the proliferation of memory T cells and advances IFN-
secretion in PHA blasts (17) and also activates memory T cells to produce the pro-inflammatory cytokine IL-17 (21, 22).
The latest member in that IL-12 cytokine family, IL-27, is a heterodimeric protein consisting of a p40-like Ebstein-Barr-Virus-induced gene 3 (EBI-3) subunit, which belongs to the hematopoietin receptor family (23), and p28, which exhibits homology to p35, p19, IL-6 and granulocyte colony-stimulating factor (G-CSF) characterized by a typical four--helix bundle cytokine structure (24). The function of IL-27 is controversially interpreted. Research shows that IL-27 acts synergistically with IL-12p70 to induce IFN-
and plays an important role in the initiation of a Th1 response (25, 26). Recent studies in mice with the defective IL-27R chain WSX-1, however, demonstrated an uncontrolled inflammatory response observed after infection with Toxoplasma gondii or Trypanosoma cruzi, emphasizing an immunoregulatory rather than an inflammatory function of this cytokine (27, 28).
Recent reports have suggested that IL-12 family members are induced after APCs were exposed to Gram-positive or Gram-negative bacteria (2931). The Gram-negative Salmonella Enteritidis belong to the group of facultative intracellular bacteria, which resist intracellular elimination (32). For a protective immunity against these pathogens an IL-12-mediated Th1 immune response is necessary that in turn depends on IFN--triggered intracellular host defense mechanisms (33, 34). The main immunostimulatory component within the cell wall of Gram-negative bacteria is LPS. Certain inbred mouse strains, such as the C57BL/10ScN strain, are highly resistant to LPS and it was shown that a natural mutation in the lps gene locus rendered the Toll-like receptor (TLR) 4 non-functional (35). The introduction of the TLR4 transgene restored the ability to respond to LPS (36).
TLRs belong to the group of PRRs which bind in a specific manner to typical microbial products also known as pathogen-associated molecular patterns. In addition to LPS, Salmonellae produce other immunostimulatory factors. Cell wall components such as lipoproteins and peptidoglycans are recognized by TLR2 in combination with TLR1 or TLR6 (37), flagellin by TLR5 (38, 39) as well as the non-methylated CpG motif of bacterial DNA recognized by TLR9 (40). Expressed by APCs, this group of receptors form the link between antigen recognition and induction of the initial immune responses (41).
This study focuses on the kinetics of mRNA expression pattern of all IL-12 family cytokines induced by the Gram-negative Salmonella Enteritidis in dependence of TLR4 and TLR2 to elucidate the central steps that lead to the induction of protective immunity. Using BMDM we also investigated the production of IL-23 and IL-12p70 proteins and describe the influence of IL-10 on their secretion.
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Methods |
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Reagents and bacteria
LPS from Salmonella abortus equi S-form and Lipid A from Salmonella Minnesota R595 were purchased from Alexis (Gruenberg, Germany). Viable Salmonella Enteritidis (vSE) and heat-killed Salmonella Enteritidis (hkSE; ade, his, SALMOVAC SE), an attenuated vaccination strain, were kindly provided by the Impfstoffwerke Dessau-Tornau (Rosslau, Germany) (43, 44). Salmonella Enteritidis were heat-killed by incubation of vSE at 60°C for 60 min. Murine IL-10 was purchased from R&D Systems (Wiesbaden, Germany). Murine IL-23 was kindly provided by DNAX Research Institute (Palo Alto, CA, USA).
BMDM
Bone marrow cells recovered from femurs of 6- to 10-week old sacrificed mice were flushed out from the bone cavities. Bone marrow cells (1 x 105 cells ml1) were cultivated for 10 days at 37°C in the presence of 30% L-conditioned medium derived from the cell line L929 (45). After a 10-day differentiation period at 37°C in humidified atmosphere with 8% CO2, macrophages were washed twice with a serum-free, high-glucose formulation of Dulbecco's modified Eagle medium and incubated overnight (5 x 105 per well) in a 24-well plate until stimulation. BMDM were stimulated with Lipid A (5 µg ml1), LPS (5 µg ml1) and hkSE or vSE (5 x 106 c.f.u. ml1). After 1, 6, 12, 24 and 48 h of stimulation, supernatants were collected and frozen at 80°C.
RNA preparation and real-time reverse transcriptionPCR
Cell lysates of stimulated and non-stimulated BMDM were prepared with RLT-lysis buffer (Qiagen, Hilden, Germany) and stored at 80°C. RNA was extracted with RNeasy® Mini Kit (Qiagen). To avoid contamination with genomic DNA, RNA samples were treated with DNase I (Promega, Mannheim, Germany) for 30 min at 37°C. DNase was inactivated using the provided stop solution and incubated at 65°C for 10 min. Oligo d(T)-primer (2.5 µM; New England Lab, Frankfurt, Germany) was supplemented to the reaction mix. The addition of RNAsin® RNase-Inhibitor (0.4 U µl1; Promega) and reverse transcription (RT) with Moloney Murine Leukemia Virus (MMLV) reverse transcriptase (1.25 U µl1; Amersham Pharmacia Biotech, Freiburg, Germany) followed immediately. According to the standard protocol, the RT reaction was performed at 37°C for 60 min. The enzyme was finally heat-inactivated at 95°C for 5 min.
Real-time PCR for different members of the IL-12 family were conducted with primers (1.0 µM) and fluorogenic probes (0.2 µM) listed in Table 1. For the investigation of the EBI-3 splice variant primers termed EBI-3_V were used. PCR was performed with Taq polymerase from Biomaster (45 U ml1; Cologne, Germany) in an iCycler (Biorad, Munich, Germany). To assess the x-fold up/down-regulation, Ct values of the housekeeping gene ß-actin were recorded for all cDNA samples. The signals for cytokine mRNA were normalized by calculating the differences (Ct) of Ctß-actin and Ctcytokine. Subsequently, the x-fold up/down-regulation was calculated by comparing stimulated with non-stimulated macrophages (
Ct) and using the formula, x-fold up/down-regulation = 2
Ct.
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Cytokine and nitric oxide measurements
Cytokine concentrations were measured by sandwich ELISA. TNF- and IL-17 were analyzed with Duo-Set® (R&D Systems) according to recommended standard protocols. IL-10 was assessed with the mAb JES5-2A5 (2.0 µg ml1; Pharmingen, Heidelberg, Germany) used as a capture antibody and for detection the biotinylated mAb SXC-1 (2.5 µg ml1; Pharmingen) were applied. IL-12p40, IL-12p70 and IL-23 were measured using the mAbs 5C3 (25 µg ml1), 48110 (2 µg ml1; R&D Systems) and 20C10 (1 µg ml1; DNAX Research Institute), respectively, as capture antibodies, and biotinylated goat anti-mouse IL-12p40-purified IgG (1 : 1000; both provided by H. Gallati, F. Hoffmann-La Roche Ltd, Basel, Switzerland) as the detection antibody. For the development with ABTS (SigmaAldrich, Taufkirchen, Germany) the biotin-labeled detection antibodies were enzyme linked with streptavidinHRP (1:3000; Southern Biotech, Birmingham, AL, USA). The measurements of ELISA at 415 nm were performed with a Spectra-max 340 ELISA reader (Molecular Devices, Munich, Germany). Nitric oxide (NO) from BMDM
supernatants were quantified with Griess reagent as described elsewhere (46).
FACS
In vitro-differentiated BMDM were harvested at day 10, pre-treated with anti-CD16/CD32 Fc block (clone 2.4G2; BD, Heidelberg, Germany) and subsequently stained with rat IgG-isotype controlFITC/PE (BD), anti-mouse F4/80FITC (Caltag Laboratories, Hamburg, Germany) or anti-mouse CD11c (clone HL3; BD). Cells were washed twice with FACS buffer (PBS/3% FCS/0.1% Na azide), fixed with PBS/1.5% (v/v) formaldehyde and subjected to FACS analysis (FACSCalibur; BD). Analyses were performed with the software CellQuestTM (BD).
IL-23 bioassay
Splenocytes were recovered from female C57BL/6 p35def/40def mice and adjusted to 4 x 106 cells ml1. After the addition of Con A (final concentration 5 µg ml1), the cell suspension was dispensed in 96-well culture plates at a final concentration of 4 x 105 cells per well. Thereafter, 100 µl of supernatants (derived from LPS- or hkSE-stimulated BMDM) to be tested, recombinant murine IL-23 at varying concentrations (positive control) and LPS or hkSE (negative controls) were added to appropriate wells and incubated at 37°C for 3 days. The IL-17 concentrations in the supernatants of the bioassay were analyzed by ELISA as outlined above.
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Results |
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After each of the three stimuli was applied, an up-regulation of mRNA was observed with a characteristic pattern. The IL-12p70 subunits p40 and p35 start a striking up-regulation at 6 h post-stimulation, that increases up to 24 h, whereas the expression of the IL-23 subunit p19 reaches the peak of induction very early (within 1 h after stimulation) and decreases afterwards. The IL-27 subunit p28 is induced with a peak at 6 h post-stimulation, whereas a minimal up-regulation of EBI-3 is apparent after LPS or Salmonella Enteritidis exposure (Fig. 1B). This indicates that a de novo mRNA synthesis of the cytokine subunits is induced in response to LPS or Salmonella starting with a characteristic early up-regulation of p19 (IL-23) at 1 h and followed by p35, p40 (IL-12p70) and p28 (IL-27) at 6 h after stimulation. In order to investigate the capability of macrophages to respond to IL-23 directly, we determined the induction of the IL-23R by real-time PCR. Six hours post-stimulation with LPS or Salmonella Enteritidis we found an up-regulation of the specific IL-23R mRNA (Fig. 1B).
Starting at different initial expression levels in resting cells, the induction of p40, p35, p19 and p28 mRNA by Salmonella Enteritidis is mediated by TLR4 but not TLR2
Since we could demonstrate an mRNA up-regulation of the IL-12 family members by Salmonella Enteritidis in BMDM, we next addressed the question which role TLR4 and TLR2 play during their induction. Therefore mRNA of stimulated BMDM
from TLR4def, TLR2def or TLR4def/TLR2def mice were compared with wild-type BMDM
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As shown in Fig. 2(A) TLR4def macrophages do not respond to the highly purified Lipid A and LPS. After stimulation with Salmonella Enteritidis, however, TLR4def cells respond with a low to moderate mRNA synthesis, and the mRNA production of p35, p19 and p28 is dramatically reduced in comparison to wild-type macrophages. The p40 mRNA production in TLR4def cells is also reduced, although the decrease is more subtle (Fig. 2A). Unexpectedly, TLR4def/TLR2def macrophages, that lack both TLRs, react stronger than the TLR4def macrophages. Unlike in TLR4def cells, the absence of TLR2 has no effect on mRNA induction of the IL-12 family members, since TLR2def cells respond similarly to wild-type cells in terms of mRNA expression of IL-12 family members.
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In stimulated macrophages there are also differences in the copy numbers between the IL-12 family members. Wild-type and TLR2def macrophages stimulated with Salmonella Enteritidis or LPS show an evident induction of p19 and p35 mRNA, but the mRNA copy numbers are much lower than those of p40 and of IL-27 (EBI-3 and p28) (Fig. 2A).
Secretion of IL-12p40, IL-10, TNF- and NO by BMDM
is chiefly mediated by TLR4
Next we investigated the secretion of cytokines, such as IL-12p40, IL-12p70, IL-23, IL-10, TNF- and the mediator molecule NO, by wild-type versus TLR4def and TLR2def BMDM
. Comparing the secretion of the p40 proteins using a p40-specific assay, there is a strong difference between TLR4def and wild-type or TLR2def cells, that is more striking than on the mRNA expression level of p40 (Fig. 3A). During the stimulation period there is a continuous increase of p40 protein level in the supernatant of the stimulated wild-type and TLR2def-macrophages, reflecting the enduring mRNA up-regulation of that subunit. Analogous to the mRNA expression levels of p40 after stimulation with Salmonella Enteritidis, the protein secretion by TLR4def/TLR2def macrophages is also stronger than by macrophages with a single TLR4 deficiency (Fig. 3A). Using specific antibodies for the heterodimeric IL-12p70 and IL-23, both cytokines were not detectable in the supernatants of stimulated and non-stimulated BMDM
of all genotypes used in the experiment, whereby the detection limit of both assays was 40 pg ml1 (data not shown). Since it was recently shown that IL-10-producing, M-CSF-differentiated macrophages, designated as macrophage type 2, are unable to secrete IL-12p70 and IL-23 (49), we determined the ability of these BMDM
to produce IL-10. The stimulated BMDM
secreted IL-10 in a TLR4-mediated manner (Fig. 3A).
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IL-10 limits the secretion of IL-23 and IL-12p70 by BMDM
To investigate the influence of IL-10 on the secretion of IL-23 and IL-12p70, we used BMDM derived from IL-10def C57BL/6 mice and compared these cells with wild-type macrophages. In contrast to macrophages with the C57BL/10 genetic background, C57BL/6 wild-type macrophages secreted measurable amounts of IL-23 and IL-12p70 after stimulation with LPS, hkSE or vSE. Nevertheless, wild-type cells differed from IL-10def macrophages in terms of a significantly reduced IL-23 and IL-12p70 secretion (Fig. 4A). The mRNA expression pattern of the p19 subunit in the IL-10def macrophages was similar to that observed in wild-type cells (data not shown). Both, IL-12p70 and IL-23, were detectable between 12 and 24 h post-stimulation. While IL-23 peaked at 24 h and started to decrease thereafter, IL-12p70 accumulated continuously. To characterize the impact of IL-10 on the synthesis of IL-23 and IL-12p70, murine IL-10 at varying concentrations was added to stimulated BMDM
derived from C57BL/6 IL-10def and wild-type mice. The addition of exogenous IL-10 resulted in a dose-dependent inhibition of the IL-23 and IL-12p70 production (Fig. 4B).
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BMDM express an EBI-3 mRNA splice variant
When EBI-3-specific conventional PCR assays were performed, an additional truncated amplicon (130 bp) was found. Analysis of organ tissues in different mouse strains (129 Sv/Ev, C57BL/6 and BALB/c) revealed that the additional fragment is present in spleen, liver and kidney of all mouse strains (data not shown). Sequence analysis of the truncated amplicon showed that the discovered fragment is a splice variant of EBI-3 mRNA, characterized by the absence of exon 2 of the full-length mRNA. Software-aided translation of the mRNA sequence into the corresponding amino acid sequence revealed an early stop codon, which probably results in a non-functional protein. Conventional PCR was used since the primers of the real-time PCR do anneal to exon 2 of EBI-3 and therefore do not detect the short splice variant. We recorded the expression pattern of the EBI-3 transcripts by analysis of equal amounts of cDNA (adjusted by ß-actin PCR) derived from LPS-, hkSE- or vSE-exposed BMDM from C57BL/10 mice. Both, the full-length transcript and the splice variant were detectable by conventional PCR before and after stimulation (Fig. 5). To investigate the expression of this EBI-3 splice variant in other APCs, we generated bone marrow-derived dendritic cells using granulocyte macrophage colony-stimulating factor (GM-CSF)-containing medium, as described elsewhere (52). Dendritic cells expressed both the full-length transcript and the splice variant before and after stimulation (data not shown) similar to BMDM
.
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Discussion |
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The heterodimeric IL-12 family members are IL-12p70 and the two new less well-defined members IL-23 and IL-27. The latter cytokines are assumed to play a crucial role in cell-mediated immune responses (54). Even if it was shown previously that a broad spectrum of bacteria is able to provoke the up-regulation and expression of various subunits within the IL-12 family (2931), a detailed kinetic analysis regarding the expression pattern of these factors has not been performed so far. The data presented here show that all members of the IL-12 family are induced by Salmonella Enteritidis albeit with varying kinetics and at different expression levels. Particularly, our studies illustrate that a quick up-regulation of p19 mRNA occurs in BMDM upon stimulation with LPS, Lipid A and Salmonella Enteritidis, whereas the mRNA syntheses for subunits that are necessary to produce IL-12p70 and IL-27 followed by a 6-h delay. This is supported by data published by Li et al., who have shown that during the early phase of EAE p19 mRNA is up-regulated in CD11b+ microglia/macrophages present in the central nervous system, whereas p40- and p35-specific mRNAs were induced later at the peak of the disease (55). These observations are probably a result of different signaling pathways that control the expression of the subunits of the IL-12 family. Analysis of the factor(s) involved in the signaling pathway leading to the early mRNA up-regulation of the p19 subunit merits further investigation. Verreck et al. argue that IL-23 is the primary pro-inflammatory type 1 cytokine produced by activated macrophages based on cell culture experiments with dendritic cells and macrophages (49). Considering the prompt up-regulation of p19 and subsequently IL-23R on macrophages in our experimental setup it is likely that IL-23 promotes an early autocrine effect on macrophages.
It is noteworthy that the mRNA data presented in this article are not limited to the sole calculation of mRNA up-regulation. We used calibrated PCR standards that allowed us to calculate the precise mRNA copy numbers in each test sample. This method provides information not only on the relative change in expression levels between treated and untreated cells but also on the precise expression levels of different factors in cells of interest. With this in mind it is remarkable that the initial expression levels of the various IL-12 family subunits vary tremendously in untreated BMDM. The IL-27 subunits EBI-3 and p28 are expressed constitutively at high levels, whereas the expression of the p35 and p19 subunits is marginal, which limit the synthesis of IL-12p70 and IL-23, respectively. In contrast, the p40 subunit shows a moderate expression level in untreated BMDM
. Starting at this moderate expression level, p40 is early accessible for protein production (
6 h) and therefore no limiting factor for IL-12p70 or IL-23. Activated BMDM
also achieve a much higher mRNA expression level for the p40 subunit than for p19 and p35. As a consequence, p40 molecules were detected in large amounts, while IL-12p70 and IL-23 were secreted at a low level in C57BL/6 (wt) macrophages and were undetectable in C57BL/10 macrophages. Studies with human myeloid cells have shown that M-CSF-differentiated macrophages produce IL-10 and suppress the secretion of IL-12p70, whereas GM-CSF-differentiated macrophages are capable of secreting IL-12p70, and as recently described, are able to produce IL-23 (49, 56). In our studies we used BMDM
grown and differentiated with L-conditioned medium, derived from L929 cells, that mainly produce M-CSF and only small amounts of GM-CSF (45, 57). BMDM
used for our studies were phenotypically different from GM-CSF-driven bone marrow cells, which others and we propose to develop into dendritic cells (52). Since we could show that the stimulated BMDM
secrete IL-10, we were interested whether IL-10-deficient macrophages produce larger amounts of p40 cytokines. We found that IL-10 limits the secretion of IL-23, whereas the mRNA expression of the p19 subunit is unchanged. This suggests that IL-10 suppresses a post-transcriptional process that influences the secretion of IL-23, but does not act on the regulation of the p19 gene expression. Although the mRNA of the p19 subunit appeared prior to p35 mRNA, both cytokines, IL-23 and IL-12p70, were detectable after the same stimulation period in wild-type as well as in IL-10-deficient macrophages (
24 h). Since it was previously shown, that IL-23 causes T memory cells to release IL-17 (21), we consider that the enhanced IL-17 release in the IL-23 bioassay was caused by bioactive IL-23 from the culture supernatants of the stimulated macrophages.
Compared with the other members of the IL-12 cytokine family, EBI-3 mRNA was detected in large amounts in resting culture macrophages. In addition, we discovered that the EBI-3 gene is transcribed in BMDM as a full-length mRNA and as a truncated splice variant. Whether this additional splice variant influences the overall EBI-3 secretion is not known. The subunit p28 also shows a high level of expression in resting cells; nonetheless, the transcription of this subunit is well induced after stimulation with Salmonella Enteritidis. In analogy to IL-12p70 and IL-23 it is possible that the smaller subunit p28 dictates the secretion of the heterodimeric protein IL-27. Considering the elevated mRNA expression levels of p28 after exposure of BMDM
to LPS or Salmonella Enteritidis one would expect an enormous output of the corresponding protein, but to our current knowledge the detection of IL-27 is technically not possible.
Here we show that the expression of the IL-12 family subunits induced by stimulation with the Gram-negative bacterium Salmonella Enteritidis is chiefly mediated by TLR4. Using highly purified LPS or Lipid A we could confirm that the mRNA up-regulation of the IL-12 family subunits is exclusively mediated by TLR4. A TLR4-mediated induction of the p19 mRNA was also demonstrated in another infection model using the Gram-negative bacterium Klebsiella pneumoniae (58).
In contrast to TLR4def macrophages, TLR2def macrophages responded like wild-type cells in terms of cytokine up-regulation and expression. This implies that LPS is the major immunostimulatory component of Salmonella that induce the IL-12 family cytokines, whereas the TLR2-mediated recognition of lipoproteins and peptidoglycans is of negligible importance and apparently provokes no IL-12 family cytokine expression when cells are exposed to Salmonella Enteritidis. A recent study has shown that only Gram-negative, but not Gram-positive non-pathogenic intestinal bacteria induced elevated p19 and p28 levels in dendritic cells (31), which demonstrates the importance of LPS-TLR4-mediated induction of p19 and p28 mRNA. Nevertheless, a TLR4-independent expression of the IL-12 cytokine family members after exposure to Salmonella Enteritidis, although at a much lesser extend, occurred in TLR4def macrophages. These observations suggest that the induction of IL-12 cytokine members is chiefly mediated by TLR4, but other PRRs different from TLR2 are most likely involved in the Salmonella Enteritidis recognition and signal triggering.
Besides, we observed differences in the response of TLR4def BMDM and macrophages that are deficient in both receptors (TLR4def/TLR2def). Double-deficient TLR4def/TLR2def macrophages always reacted stronger than the single-deficient TLR4def cells. The reasons for that observation are not yet investigated and might be the consequence of a higher amount of accessible intracellular adaptor proteins in cells with two non-functional receptors or a compensatory up-regulation of other PRRs.
In summary, we have demonstrated a TLR4-mediated induction of mRNA of the IL-12 family members by Salmonella Enteritidis characterized by differing kinetics and varying expression levels in resting and stimulated BMDM. Macrophages show high-level production of mRNA for the IL-27 subunits, whereas the secretion of IL-23 and IL-12p70 is restricted by a moderate mRNA expression and is negatively regulated by IL-10. The quick and transient up-regulation of the p19 mRNA suggests a time-limited secretion of IL-23 cytokine after microbial activation of macrophages. Since the specific IL-23R was induced in macrophages after LPS stimulation (20), this implies that IL-23 released by macrophages probably unfolds an autocrine effect.
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Acknowledgements |
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Abbreviations |
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APC | antigen-presenting cell |
BMDM![]() | bone marrow-derived macrophages |
CSF | colony-stimulating factor |
EAE | experimental autoimmune encephalomyelitis |
EBI-3 | Ebstein-Barr-Virus-induced gene 3 |
GM-CSF | granulocyte macrophage colony-stimulating factor |
hkSE | heat-killed Salmonella Enteritidis |
M-CSF | macrophage colony-stimulating factor |
NO | nitric oxide |
PRR | pattern recognition receptor |
RT | reverse transcription |
TLR | Toll-like receptor |
TNF | tumor necrosis factor |
vSE | viable Salmonella Enteritidis |
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Notes |
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Received 12 November 2004, accepted 22 February 2005.
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References |
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