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International Immunology, Vol. 14, No. 10, pp. 1203-1213, October 2002
© 2002 Japanese Society for Immunology

Global reprogramming of dendritic cells in response to a concerted action of inflammatory mediators

Malin Lindstedt1, Bengt Johansson-Lindbom1 and Carl A. K. Borrebaeck1

1 Department of Immunotechnology, Lund University, PO Box 7031, 220 07 Sweden

Correspondence to: C. A. K. Borrebaeck; E-mail: carl.borrebaeck{at}immun.lth.se
Transmitting editor: P. W. Kincade


   Abstract
Top
 Abstract
Introduction
 Methods
 Results and discussion
 Conclusion
 References
 
Maturation of dendritic cells (DC) serves a deterministic role in the link between innate and adaptive immunity, constituting a checkpoint with regard to whether responses from the lymphocyte compartment shall be raised and what class of response is needed to protect the host against invading pathogens. Since DC have not been shown to possess mechanisms such as gene recombination or somatic mutation for generating a diverse repertoire of antigen-recognition receptors, it is unlikely that these leukocytes can intrinsically respond to all conceivable molecules present in our environment. In the present study, we have therefore determined how mediators of the inflammatory response regulate global gene transcription in DC. The data represent an extensive and time-ordered reprogramming of the DC during their course of maturation, involving genes encoding proteins that regulate responses of both innate cells and lymphocytes. This transcriptional reorganization may reflect the effect of in vivo released inflammatory mediators induced by endogenous or pathogenic stimulation.

Keywords: cellular activation, dendritic cell, immunomodulator, inflammation, inflammatory mediator


   Introduction
Top
 Abstract
 Introduction
Methods
 Results and discussion
 Conclusion
 References
 
Dendritic cells (DC) are bone marrow-derived and highly specialized antigen-presenting cells (APC) that can activate naive T cells and hence initiate primary immune responses (1). Although currently recognized as a heterogeneous population of cells, possibly encompassing several subsets or lineages (2–4), DC commonly display discrete functional states segregated both in time and space (5–8). The transition between these states constitutes a crucial event for induction of the adaptive immune response, and involves a switch in DC function and localization, from efficiently acquiring antigens at sites of, for example, inflammation to presenting these in an immunogenic context for T cells localized to the paracortex of secondary lymphoid organs (1). DC may also play a crucial role in shaping the class of response being elicited. Potentially, this property of DC is conferred by either the recruitment of separate subsets of DC with distinct properties (9,10) or by an intrinsic capability of one and the same subset to differentially respond to distinct maturational stimuli (11–13). Furthermore, the kinetics of DC activation may also contribute as an important parameter to the outcome of T cell differentiation (14,15). Accordingly, monocyte-derived DC exposed to lipopolysaccharide (LPS) for a short period of time produce large quantities of IL-12 and consequently drive Th1 development, whereas longer periods of LPS exposure render the same cells deficient in IL-12 production. Hence, T cells primed on DC subjected to sustained maturation preferentially develop into Th2 cells or quiescent cells apparently lacking effector functions (15). Consistent with the transient release of IL-12 from human DC in vitro, Toxoplasma gondii tachyzoites-induced IL-12 production in murine CD8{alpha}+ DC in vivo is also very short-lived (16).

The time-ordered and reciprocal regulation of separate genes during DC activation clearly demonstrates the need to investigate the expression, kinetics and function of each of these genes in relation to the global pattern of genes being regulated in response to a particular provocation (17). In this report, we compare the transcriptional profiles of human monocyte-derived DC at 0, 8 and 48 h after exposure to inflammatory maturational stimuli, consisting of monocyte-conditioned medium (MCM) supplemented with additional tumor necrosis factor (TNF)-{alpha} and IL-1ß (MCM+). For this purpose we have applied a high-density oligonucleotide microarray technique, monitoring 12,500 genes in parallel. We demonstrate that DC maturation provoked by this inflammatory stimuli causes dramatic changes in gene expression and, furthermore, that the process is highly dynamic. Thus, maturation in vivo might be a result of the microenvironment of inflamed tissue (18), and our results demonstrate a possibility for pathogens to indirectly trigger DC maturation and hence mobilize the adaptive immune response by provoking inflammation.


   Methods
Top
 Abstract
 Introduction
 Methods
Results and discussion
 Conclusion
 References
 
Reagents and cytokines
Cell culture medium RPMI 1640 was supplemented with 10% (v/v) FBS (Gibco, Grand Island, NY), 50 µg/ml gentamicin, 2 mM L-glutamine and 1% (v/v) non-essential amino acids (Life Technologies, Rockville, MD), and is referred to below as R10 complete medium. Recombinant human granulocyte macrophage colony stimulating medium (GM-CSF; Leukomax) was obtained from Sandoz (Schering-Plough, Innishannon, Ireland). rhIL-1ß, rhTNF-{alpha} and rhIL-4 were purchased from R & D Systems (Minneapolis, MN), and LPS was purchased from Sigma-Aldrich (St Louis, MO). MCM was obtained by culturing monocytes in R10 complete medium for 24 h at a cell density of 2 x 106 cells/ml, after which the cells were excluded from the medium.

Cell surface antigen analysis
FACS analysis was performed on a FACScan (Becton Dickinson, San Jose, CA), using CellQuest analysis software. PBS (Ca/Mg-free; Gibco) containing 1% BSA (Sigma-Aldrich) was used in all cell labeling and washing steps. Cells were stained with various antibodies, diluted to the optimal staining concentration, for 30 min on ice, washed and resuspended in PBS/BSA. Data analysis was based on examination of 1 x 104 events/sample, and gates were set to exclude debris and non-viable cells. FITC-conjugated anti-CD40 and phycoerythrin (PE)-conjugated anti-HLA-DR and anti-OX40 mAb were purchased from Becton Dickinson (Mountain View, CA). FITC-conjugated anti-CD1a and anti-CD11c as well as PE-conjugated anti-CD14, anti-CD25 and rabbit-anti-mouse Ig mAb were obtained from Dako (Glostrup, Denmark). FITC-conjugated anti-CD80 and anti-CD83 mAb were purchased from Immunotech (Marseille, France). Anti-CD86 mAb (Bu63), a generous gift from John Pound (Birmingham, UK), was purified from ascites fluid by ion-exchange chromatography on SP-Sepharose (Pharmacia Biotech, Uppsala, Sweden) and conjugated to FITC according to standard protocols. Mouse anti-CCR7, and biotin–anti-mouse IgM were obtained from PharMingen (San Diego, CA). Mouse anti-CD137 was obtained from Biosource (Camarillo, CA). Anti-DC-LAMP was a generous gift from S. Lebeque (Shering-Plough, Lyon, France). DC-LAMP and CCR7 expression was visualized by PE-conjugated streptavidin from Dako. Appropriate isotype-matched non-specific controls were used in all experiments to determine the levels of background staining.

Giemsa staining
To visualize the morphology of monocyte-derived DC, cytospins were prepared from cell samples during the differentiation. Cells were centrifuged in R10 complete medium (5 min, 60 g) onto microscope slides and air dried overnight. Giemsa stain (Sigma-Aldrich) was added to the slides for 15 min, which were subsequently washed with distilled water.

Generation of monocyte-derived DC
Peripheral blood mononuclear cells were isolated from leukocyte-enriched buffycoats, obtained from Lund University Hospital (Lund, Sweden) by Ficoll-Paque (Pharmacia, Piscataway, NJ) density gradient centrifugation. T lymphocytes were depleted by rosetting with sheep erythrocytes treated with neuraminidase (Sigma-Aldrich). CD14+ monocytes were purified by magnetic cell sorting, using microbead-conjugated anti-CD14 antibodies (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany) and magnetic cell separation columns (Miltenyi Biotec), resulting in >97% purity of CD14+ cells. To generate DC, CD14+ monocytes were cultured (5 x 105 cells/ml) for 7 days in R10 complete medium supplemented with rhGM-CSF (300 ng/ml) and rhIL-4 (100 ng/ml). Half of the amount of medium with cytokines was exchanged every 2–3 days. After 7 days of culture, the DC were stimulated with rhIL-1ß (10 ng/ml), rhTNF-{alpha} (10 ng/ml), LPS (1 µg/ml) or MCM+ [rhIL-1ß (10 ng/ml) and TNF-{alpha} (10 ng/ml)]. Different batches of MCM derived from separate donors were utilized to reduce the risk of biased results. Cell samples of mature DC were analyzed at 8, 24 and 48 h after stimulation.

Preparation of cRNA and gene chip hybridization
Total RNA was isolated from monocytes and from immature monocyte-derived DC cultured for 3 and 7 days with GM-CSF and IL-4. RNA was also isolated from mature monocyte-derived DC at 8 h and 2 days after addition of maturation stimuli. Cell samples were lyzed in TRIzol Reagent (Gibco) and the total RNA was used to generate labeled cRNA probes. Preparation of cRNA, hybridization and scanning of the HuGeneFL Arrays were performed according to the manufacturer’s protocol (Affymetrix, Santa Clara, CA). Briefly, cDNA was generated from total RNA (5 µg) using the Superscript Choice system (Gibco) with a dT24 primer containing a T7 RNA promoter site. cDNA was converted to labeled cRNA by an in vitro transcription reaction (ENZO, Farmingdale, NY) with biotin-labeled ribonucleotides and T7 RNA polymerase. Thereafter, the cRNA was purified using a RNeasy Mini kit (Qiagen, Valencia, CA) and the yield controlled with spectrophotometry. cRNA (20 µg) was fragmented in 40 mM Tris–acetate, pH 8.1, 100 mM KOAc and 30 mM MgOAc at 94°C for 35 min. The fragmentation step was controlled by agarose gel electrophoresis. The RNA (15 µg) was used in a 200 µl hybridization cocktail [0.1 mg/ml herring sperm DNA (Promega, SDS, Santa Cruz, CA), 100 mM MES (Sigma-Aldrich), 1 M sodium chloride, 20 mM EDTA, 0.5 mg/ml BSA, 0.01% Tween 20, 0.05 µg/µl control primer B2, pH 6.5–6.7). A number of hybridization controls were added to the cocktail, such as the Escherichia coli genes BioB (150 pM), BioC (500 pM) and BioD (2.5 nM), and the P1 bacteriphage gene Cre (10 nM). The control RNAs were used to compare the hybridization efficiency between arrays. Before hybridization, the cRNA samples were heated (99°C, 5 min), equilibrated (45°C, 5 min) and clarified by centrifugation (14,000 g, 5 min at room temperature). The samples were then hybridized to the Human Genome U95A arrays at 45°C for 16 h by rotation (60 r.p.m.) in an oven. The arrays were then washed, stained with streptavidin–PE (Molecular Probes, Eugene, OR), washed again and scanned with a GeneArray Scanner (Affymetrix). The fluorescence intensity was analyzed using Microarray Suite 4.0 software (Affymetrix) which includes algorithms that determine whether a gene is absent, marginally present or present in the sample. The arrays were normalized based on average intensity on the chip and further data analysis was performed with GeneSpring 4.0 software (Silicon Genetics, Redwood City, CA).

cDNA synthesis and RT-PCR analysis
cDNA synthesis was performed from the total RNA, used for the gene chip hybridizations. Total RNA (2.5 µg) from each cell sample was mixed with 2 ng oligo(dT) (Promega, Madison, WI) and incubated for 10 min at 70°C. The template was added to a mixture of 5 x first strand buffer, 0.1 M DTT (Gibco) and 10 mM dNTP (Pharmacia) for 2 min at 37°C. The mixture was then incubated with 40 U RNasin and 500 U Superscript II reverse transcriptase (Gibco) for 1 h at 42°C, and subsequently denatured at 75°C for 10 min. PCR reactions were performed in 50 µl containing 2.5 U AmpliTaq DNA polymerase, PCR buffer (Roche Diagnostics, Mannheim, Germany), 20 µM 3' and 5' CD137 primers (5'-TAGTAGCCACTCTGTTGCTGGT-3' and 5'-CCTCTCCTTCGTCCCATTCACA-3') and 1.25 mM dNTP (Roche Diagnostics). Thirty-eight cycles were performed (94°C for 1 min, 65°C for 1 min and 72°C for 2 min) in a Perkin-Elmer GeneAmp PCR system 2400, generating a 440-bp product. Human tonsillar DC were isolated as described previously (19).


   Results and discussion
Top
 Abstract
 Introduction
 Methods
 Results and discussion
Conclusion
 References
 
Maturation of monocyte-derived DC in response to inflammatory stimuli
Monocyte-derived DC were obtained by culturing purified blood monocytes for 7 days in GM-CSF and IL-4 (20). These immature DC were consistently >91% CD14–CD1a+ and <2% CD14+. We then compared inflammatory stimuli and LPS for their relative ability to induce strong and sustained maturational reprogramming of the immature monocyte-derived DC. For this purpose we used medium from cultured plastic-adherent monocytes. Even though MCM in several studies has been shown to support DC maturation with high efficacy (21–23), such medium has been prepared by stimulation of monocytes with plastic-adsorbed human IgG. This particular stimulation may specifically elicit the production of soluble mediators as a result of cross-linking of Fc{gamma} receptors (24) and it may therefore not be representative for in vivo primary immune responses. For this reason we omitted the human IgG and instead relied on factors being released solely as a result of the activation, occurring when the cells adhere to plastic (25,26). Immature DC, exposed to a MCM+ cocktail, up-regulated surface CD86 during the 7-day culture period, as did stimulation with LPS or TNF-{alpha}/IL-1ß (Fig. 1A). If the DC were first cultured for 2 days with either LPS, TNF-{alpha}/IL-1ß, MCM or MCM+ and then cultivated for an additional 5 days after removal of these stimuli, a substantial proportion of the cells that initially were exposed to the MCM reverted to a CD14+ phenotype (Fig. 1B). This is indicative of a transient and not terminal DC differentiation (27), and demonstrates that the process consists of different maturational thresholds. However, cells first exposed to MCM+ retained their CD14– phenotype to a higher extent even if the MCM+ was depleted from the cultures (Fig. 1B). Furthermore, since monocyte-derived factors in MCM+ did not inhibit the maturation and up-regulation of CD86 (Fig. 1A), we decided to use the MCM+ in further studies. This decision was also partially based on the fact that soluble factors others than TNF-{alpha} and IL-1ß are produced during inflammation (18), and inflammatory mediators released from resident macrophages, for example, and infiltrating leukocytes will clearly participate in this process (28–30).



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  		Fig. 1. Expression of surface CD86 and CD14 by DC in response to various activation stimuli. (A) Percentage CD86+CD14– DC 2 and 7 days after induction of maturation by LPS, TNF-{alpha} and IL-1ß, MCM or MCM+. (B) Percentage of cells that reverted to a CD14+ phenotype after 2 days of culture with maturation stimuli LPS, TNF-{alpha}/IL-1ß, MCM or MCM+ and then an additional 5 days of culture after removal of stimuli.

 
Kinetics of monocyte-derived DC maturation and high-density microarray analysis
The temporal response of monocyte-derived DC to the MCM+ was thereafter studied using flow cytometry and mAb to defined differentiation markers (Fig. 2A). Consistent with previous reports (1), surface antigens which in vivo are strictly confined to T cell zone-localized DC subsets, i.e. CD83, CD86, DC-LAMP and CCR7, exhibited slow kinetics for induction. Although strong expression of these molecules could be visualized after a culture period of 24–48 h in MCM+, at earlier time points their densities ranged from low to not detectable. In addition, expression levels of CCR7 and DC-LAMP, for example, were progressively increasing during the entire 48 h course of maturation, demonstrating that a plateau in expression intensity is not reached for these proteins unless the cells are exposed to MCM+ for an extended period of time (>=48 h). To identify early events of the DC maturation process, we examined morphological alterations over time. Despite exhibiting a phenotype fairly similar to the appearance of immature DC, cells activated for only 8 h acquired an irregular shape with veiled processes typical of a more mature DC (Fig. 2B). Since these DC not yet have reached the T cell zone-associated mature phenotype, the evident morphological signs of maturational progression suggest that the cells may reflect the early and peripheral gene expression, which takes place in DC in inflamed peripheral tissues (31,32). In particular, this assumption is supported by the failure to detect any CCR7 by FACS analysis, since CCR7 expression is required for migration towards secondary lymphoid organs where the ligands ELC and SLC are produced (33). Taken together, DC matured for 8 and 48 h were chosen as representatives for early and late DC maturation respectively, and further compared at the level of mRNA with their immature counterparts.



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  		Fig. 2. Surface phenotype and morphology of monocyte-derived DC. (A) CD14+ monocytes were cultured for 7 days with GM-CSF and IL-4 to acquire the immature DC phenotype (iDC) and for an additional 48 h after addition of MCM+ to generate the mature DC (mDC). Cell surface proteins expressed by immature DC after 7 days, and mature DC after 8 and 48 h of stimulation were studied with FACS. The gray histograms represent staining with specific FITC- and PE-conjugated antibodies, and the white histograms represent the isotype-matched controls. (B) Giemsa staining of cytospins prepared from immature and mature DC.

 
Microarray technology (34) was used to assess the transcriptional activity of ~12,500 genes simultaneously during DC maturation. Labeled hybridization samples were prepared in duplicates from mRNA obtained from monocyte-derived DC exposed to the MCM+ for 0, 8 and 48 h respectively. For each time point, samples from two donors were run in parallel. Of the total number of probe sets on the array, 42–50% were denoted as ‘Present’ on each individual chip, which means that 5300–6300 genes were expressed in each cell type. These genes were clustered using a K-means clustering algorithm (35) and four separate clusters, representing different kinetics of gene-expression were defined (Fig. 3). These clusters include genes being up-regulated in the DC after 8 h of maturation (Fig. 3A), genes exhibiting a fast induction and sustained expression (Fig. 3B), and genes being progressively up-regulated (Fig. 3C) or down-regulated (Fig. 3D) during the overall course of maturation. Genes exhibiting >=2-fold change in average intensity between two separate measurements were considered to be significantly up- or down-regulated and are shown in Tables 1–4 (Fig. 3).



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  		Fig. 3. Patterns of gene expression during DC maturation based on K-means clustering analysis. Monocytes were cultured with GM-CSF and IL-4, and cells samples were collected after 7 days of DC differentiation. The immature DC were stimulated at day 7 and cultivated for an additional 2 days with MCM+. Cell samples were extracted at 0, 8 and 48 h after induction of maturation. Each line represents expression of one gene. Clusters with different kinetics of gene expression include genes up-regulated at 8 h (A), 8 and 48 h (B), and 48 h (C) of maturation, and genes down-regulated during the course of maturation (D).

 

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  		Table 1. Genes up-regulated in DC stimulated for 8 h as compared to DC stimulated for 48 h (cluster A, Fig. 3)
 
Inflammatory stimuli elicit a comprehensive and temporal re-programming of the monocyte-derived DC transcriptome
In agreement with the present notion of separate transcriptional phases during LPS induced DC maturation (12,15,32), for example, the endogenous factors of the MCM+ also caused DC to transcribe genes in a highly time-ordered fashion. Thus, a great number of genes were strongly but only transiently induced (Fig. 3A and Table 1), as illustrated by up-regulation of a broad panel of inflammatory chemokines at the 8 h time point, including IL-8, MIP-1{alpha}, MIP-1ß, RANTES, MCP-2, MIP-3{alpha}, GRO-{alpha}/MGSA, GRO-ß, I-309, IP-10 and I-TAC. All these transcripts were present at considerably lower levels after 48 h of maturation, demonstrating the temporal nature of their expression. This comprehensive array of chemotactic mediators also demonstrates a diverse leukocyte specificity (36). Genes with fast activation kinetics also included the pro-inflammatory cytokines IL-1ß, TNF-{alpha} and IL-6, as well as IL-7 and GM-CSF. Moreover, in agreement with a transient release of IL-12 in response to LPS (15), the p40 transcript of IL-12 was also highly induced after 8 h of maturation and exhibited a 20-fold decrease in intensity after sustained activation. Also, the p35 subunit of IL-12 conformed to the transient up-regulation (data not shown), indicating that IL-12 is produced in response to endogenous inflammatory stimuli (37), promoting a Th1 differentiation. However, the tremendous increase in mRNA for I-309 (>150-fold increase), a chemokine that preferentially recruits Th2 cells (38), indicates a different but yet relevant role for monocyte-derived DC in also amplifying Th2 responses at an early stage in vivo. In addition, two other chemokines which also appear to preferentially attract Th2 cells, MDC (39) and TARC (40), were transcribed at high levels after 8 h of maturation (as judged by strong average intensity values), although they were also present at equally high levels in the immature population of the DC (data not shown). Memory B cells may also migrate toward peripheral tissues in response to the DC-produced MIP-3{alpha}, since they express high levels of the MIP-3{alpha} receptor CCR6 (41). In accordance with the transient but massive expression of MIP-3{alpha} mRNA in response to MCM+ (Table 1), production of this chemokine in inflamed tonsil is strictly confined to epithelial crypts, which is the site of antigen entry and where memory B cells indeed co-localize with immature DC (42). The MCM+ also induced transcription of the OX40 gene which is a T cell-associated molecule involved in signaling for the re-localization of CD4+ T cells from T cell zones to the follicles (43). On the other hand, OX40-mediated triggering of B cells expressing OX40L has been reported to increase the IgG secretion after immunization with a haptenated protein (44). Taken together, these data reinforce the remarkable potential of DC to attract and influence a wide spectrum of leukocytes, including cells of the adaptive immune response, at an early phase of their maturation. Also the kinetics of CCR7 regulation is in line with this assumption, since this transcript was only 5-fold up-regulated after 8 h of activation as compared to a 24-fold increase after a 48 h exposure to MCM+ (Table 3). Furthermore, irrespective of exerting important roles in innate or adaptive immunity, the diversity of these mRNA species distinctly shows that the concerted action of pro-inflammatory components can substitute for a limited capacity of certain pathogens to trigger DC maturation, provided that these microbes provoke inflammation. This is exemplified by the fact that a substantial number of the genes listed in Tables 1–4 was recently reported to be differentially regulated in DC challenged with pathogens, such as E. coli, influenza and Candida albicans (12). Of the differentially regulated genes, as many as 50% were also found in DC stimulated with MCM+, implying that these genes may be expressed as an effect of autocrine release of inflammatory mediators. In addition to these differentially expressed genes, E. coli, influenza and C. albicans induced a common set of highly regulated genes, which is regarded as a core DC response to pathogenic stimulation. A comparison between this pathogen-induced core response with the inflammation-mediated maturation reveals a similar expression pattern. Up to 70% of the pathogen-induced transcripts are also found in the DC stimulated with MCM+, suggesting that a majority of the core response genes can be regarded as general activation genes, such as IL-1ß, IL-6 and TNF-{alpha} (Table 1), which may act in an autocrine fashion to enhance the activation. One family of genes that clearly differed between the pathogen- and inflammation-induced transcripts is the antiviral genes, encoding type 1 IFN, which was produced by influenza-stimulated DC but not by MCM+ matured DC. High production of IFN-{alpha} in response to viral infection has been shown to be a feature of TLR9+ plasmacytoid pre-DC (8). This indicates that viral infection stimulates DC to a highly specific response.


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  		Table 3. Genes up-regulated in DC stimulated for 48 h as compared to DC stimulated for 8 h (cluster C, Fig. 3)
 
Transcripts expressed with fairly equal intensity during both the late and early stage of the maturation comprise many of the genes involved in NF-{kappa}B signal transduction, such as TRAF1, NF-{kappa}B (p150), NF-{kappa}B2 (p49/p100) and I{kappa}B (45) (Table 2). Although DC to a great extent lose their capacity to respond to external stimuli after prolonged activation, the sustained transcription of NF-{kappa}B family members may provide an explanation for how DC retain responsiveness to certain stimuli during the entire course of their maturation. For example, prolonged TNF-{alpha}/IL-1ß-driven DC maturation prevents subsequent CD40L/IFN-{gamma}-dependent induction of IL-12 p70, but not CD40L-dependent production of IL-6 (14).


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  		Table 2. Genes up-regulated in DC stimulated for 8 and 48 h (cluster B, Fig. 3)
 
The expression of mRNA-encoding molecules involved in co-stimulation of both T and B cells increased during the MCM+-induced maturation. CD86, OX40L, RANK and OX2 mRNA expression increased progressively (Table 3), whereas transcripts for CD40, OX40 and CD137L (4-1BBL) reached their peak of expression at 8 h after stimulation (Table 1). Furthermore, increased levels of mRNA encoding CD80 and CD137 (4-1BB) remained elevated also after sustained exposure to the MCM+ (Table 2). Even though the literature is full of studies describing functional properties of most of these molecules in relation to DC–T cell interactions (46), some aspects of their appearance at the mRNA level deserve comments. The induction of mRNA for receptor–ligand pairs in the monocyte-derived DC implies a bilateral function for DC interacting with both B and T lymphocytes, as was discussed above for the OX40–OX40L. Transcripts for CD137–CD137L (a pair of the TNF–TNF receptor superfamilies) were also present in the MCM+-matured DC. CD137L is expressed on various APC, and provides co-stimulation for naive CD4+ T cell proliferation and IL-2 secretion, either alone (47) or in synergy with CD28 (48). CD137-mediated signaling can with equal efficacy also trigger CD8+ T cell proliferation and effector functions (49), including cytotoxic T lymphocyte activity (50) and tumor rejection (51). Whereas DC expressing CD137L most likely play an important role in the initiation of several of these T cell responses, the co-expression of CD137 suggests novel functions for DC in the regulation of other APC. Engagement of the receptor–ligand pair has indeed been shown to transmit a reverse signal through CD137L, which had a strong impact on functions of both monocytes (52) and, in particular, of B cells (53).

Finally, Table 4 lists genes which were gradually down-regulated when monocyte-derived DC where exposed to MCM+. Consistent with the reduced capability of mature DC to acquire and process antigens (1), several of the genes in this cluster encode proteins which are involved in antigen capture and processing. Thus the levels of mRNA encoding Fc and complement receptors, as well as aquaporin 3 facilitating macropinocytosis (54), were decreased. Transcripts for HLA-DM catalyzing peptide loading to MHC were also reduced (55). Parallel to these events, several molecules involved in the adhesion to cells and extracellular matrix in peripheral tissues lost their expression of mRNA. Examples of such genes are E-cadherin (56), N-cadherin (57), CD18 (58), CD49e (56) and vinculin (59). These were instead replaced by transcripts coding for a new panel of adhesion proteins, including CD44, CD48, CD54 and CD58, which can facilitate DC homing to lymph nodes or promote physical interaction between the DC and lymphocytes (56,60,61).


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  		Table 4. Genes down-regulated in maturing DC (cluster D, Fig. 3)
 
Many genes not described previously in DC were found in the four clusters, such as putative chemokine receptor HM-74, OX40, macrophage lectin 2, C-type lectin, MARCKS and nuclear orphan receptor (MINOR).

Confirmation of OX40 and CD137 expression
Several reports have confirmed a direct functional role for DC in the regulation of B cell responses (19,62,63). Even though most of these studies have established that the DC-mediated effects on B cell proliferation, Ig class switching and secretion were preferentially mediated by soluble factors, they have not been performed with antigen-specific systems. Using immune complexes formed with hen egg lysozyme (HEL), it was recently demonstrated that that HEL-specific B cells acquired the HEL antigen from APC upon cognate interaction and synapse formation (64). A functional role for physical contact between B cells and DC has also been demonstrated by MacPherson et al. (65). To further substantiate the possible role DC-expressed OX40 and CD137 may have in the DC cell interaction, we confirmed their expression at the protein level using specific mAb and flow cytometry (Fig. 4). The cell surface expression of OX40 was evident after 48 h of activation (Fig. 4B), which is somewhat later compared to mRNA that peaked after 8 h (Fig. 4A). However, intracellular staining revealed an up-regulation of the protein also at earlier time points. In addition to demonstrating the expression of CD137 on matured DC, we also confirmed the presence of the corresponding mRNA in these cells and also in freshly sorted CD40high tonsil DC (19), using RT-PCR (Fig. 5).



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  		Fig. 4. Changes in gene and protein expression of DC-LAMP, OX40 and CD137 during DC activation. (A) The level of gene expression, based on the average intensity on the gene chips, throughout the differentiation and activation of DC. Immature DC (day 7) were stimulated with MCM+, and samples were collected at 8 and 48 h after induced maturation. (B) Cell surface and intracellular expression in immature DC after 7 days in culture, and mature DC after 8, 24 and 48 h of stimulation were studied with FACS. MFIs were calculated by subtracting the intensities of appropriate isotype-matched controls. In both graphs, the first y-axis presents the values for DC-LAMP and OX40, and the second y-axis presents the values for CD137.

 


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  		Fig. 5. CD137 transcripts expressed by matured monocyte-derived DC and tonsillar DC. RT-PCR analysis was performed on freshly isolated monocytes (Mo), monocyte-derived DC stimulated with MCM+ for 48 h (mDC) and freshly sorted CD40high tonsil DC (tDC). GAPDH mRNA levels were used to standardize total mRNA levels in the cell populations.

 

   Conclusion
Top
 Abstract
 Introduction
 Methods
 Results and discussion
 Conclusion
References
 
Several pathogen-derived molecules have been identified as potent inducers of DC maturation, and chemically diverse molecules such as LPS and double-stranded RNA provoke distinct responses by DC in vitro (12). During physiological conditions, however, a more general route for triggering DC maturation resides in the mediators released from inflamed tissues. Resident tissue macrophages, epidermal keratinocytes, endothelial cells, stromal cells and infiltrating mononuclear cells are all implicated in the inflammatory response, and constitute sources of lipid mediators and pro-inflammatory cytokines, for example (66–68). Tissue damage and necrotic cell death may be the very initial events which provoke the release of these entities, further emphasizing the importance of endogenously derived stimuli in the in vivo process of DC maturation (18). In the present study, we have therefore studied the DC differentiation process in response to pro-inflammatory cytokines TNF-{alpha} and IL-1ß plus entities released by plastic-adherent monocytes, partially reflecting the inflammatory response that is raised against various invading pathogens at the site of their entry. This assumption is supported by several reports demonstrating the participation of TNF-{alpha} and IL-1ß during an early phase of inflammation (68,69). Furthermore, although in vitro experiments have provided evidence for a poor capacity of individual pro-inflammatory cytokines such as TNF-{alpha} in driving terminal DC maturation (26), our data and previous reports (20,21) demonstrate that the concerted action of several inflammatory mediators clearly achieves this purpose.


   Acknowledgements
 
This work was supported by a grant from the European Commission (QLK3-CT-2000-00270) and Vårdalstiftelsen. We would like to thank Ann-Charlotte Ohlsson for her expert technical assistance.


   Abbreviations
 
APC—antigen-presenting cell

DC—dendritic cell

GM-CSF—granulocyte macrophage colony stimulating factor

HEL—hen egg lysozyme

LPS—lipopolysaccharide

MCM—monocyte-conditioned medium

MCM+—MCM with additional TNF-{alpha} and IL-1ß

PE—phycoerythrin

TNF—tumor necrosis factor


   References
Top
 Abstract
 Introduction
 Methods
 Results and discussion
 Conclusion
 References
 

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