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Blood, 1 August 2005, Vol. 106, No. 3, pp. 946-955. Prepublished online as a Blood First Edition Paper on April 26, 2005; DOI 10.1182/blood-2004-08-3228.
IMMUNOBIOLOGY Osteopontin functionally activates dendritic cells and induces their differentiation toward a Th1-polarizing phenotypeFrom the Department of Dermatology and Allergology, University of Ulm, Germany; the Department of Dermatology, University of Freiburg, Germany; the Biozentrum Basel, University of Basel, Switzerland; the Institute for Genetic Medicine, Hokkaido University, Japan; and the Department of Dermatology, Venerology and Allergology, University of Leipzig, Germany.
Osteopontin (OPN) has been shown to have T helper 1 (Th1) cytokine functions in cell-mediated immunity. Deficiency of OPN is linked to a reduced Th1 immune response in autoimmunity, infectious disease, and delayed-type allergy. Dendritic cells (DCs) are central for the induction of T-cell–mediated immunity, when initially flexible DCs are instructed by priming signals and tissue-derived factors to adopt Th1, Th2, or regulatory T-cell–inducing phenotypes. Although OPN influences the cytokine secretion of T cells and macrophages, its effects on DC polarization remain an important missing link in the understanding of OPN functions in Th1 immunity. Here we demonstrate that OPN promotes the emigration of human DCs from the epidermis and functionally activates myeloid-type DCs, augmenting their expression of HLA-DR, costimulatory, and adhesion molecules. OPN induces their Th1-promoting tumor necrosis factor  (TNF-) and interleukin-12 (IL-12) secretion, and enhances their allostimulatory capacity. In mixed lymphocyte reactions (MLRs), OPN stimulates IL-12 secretion by DCs, inducing elevated interferon- (IFN-) production by T cells. Naive Th cells stimulated by OPN-activated DCs show a Th1-polarized cytokine production. Our findings identify OPN as an important tissue-derived factor that DCs encounter when traveling from peripheral sites of activation to secondary lymphatic organs, which induces DC maturation toward a Th1-promoting phenotype.
Osteopontin (OPN) is a secreted phosphoprotein that contains an integrin-binding arginine-glycine-aspartate sequence (RGD) motif. OPN has proinflammatory cytokine and chemokine functions in cell-mediated immunity.1-4 A number of studies have demonstrated that OPN critically contributes to the development of T helper 1 (Th1)–mediated immunity and disease.5,6 Among other reports, OPN-deficient mice develop disseminated infection and have a delayed ability to clear disease when infected with Mycobacterium bovis (BCG).7 In murine models of autoimmune encephalomyelitis, a model of human multiple sclerosis that critically depends upon the balance of Th-shaping cytokines such as interleukin-10 (IL-10) and IL-12, OPN-deficient mice develop milder disease.8,9 Corneal infection of OPN-deficient mice with herpes simplex virus 1 (HSV-1) is followed by a reduced delayedtype hypersensitivity to HSV.6 We have shown that OPN-deficient mice have an impaired allergic contact hypersensitivity (CHS) response against trinitrochlorobenzene, which is accompanied by a reduced ability to attract dendritic cells (DCs) from the periphery into draining lymph nodes.10 DCs are the most potent antigen-presenting cells (APCs). Their function and polarizing capacities are decisive for the outcome of Th-mediated immunity.11-13 Although, it is known that OPN plays a central role for the initiation of Th1-mediated immune responses, it is unknown whether it is involved in DC instruction to induce Th1-mediated responses. In their immature state, DCs are situated in peripheral nonlymphoid tissues.14,15 For example, Langerhans cells (LCs) are located in the epidermis. Upon stimulation, LCs/DCs undergo a maturation process resulting in the down-regulation of their antigen uptake and processing capacity; up-regulation of major histocompatibility complex (MHC) II and costimulatory molecules; and a switch in their expression pattern of chemokines and adhesion molecules.14,15 As a consequence of this reprogramming, DCs migrate into lymphoid organs.16,17 In the T-cell zone of lymph nodes, they function as APCs, which prime naive antigen-specific T cells and drive their differentiation toward Th1, Th2, or regulatory T cells.11-13
The initial activation stimulus in concert with tissue environmental factors encountered by migrating DCs instruct DCs to polarize toward a phenotype that initiates Th1, Th2, or regulatory T effector cells.11,12,18,19 Numerous viral and microbial factors, among them lipopolysaccharide (LPS), Staphylococcus aureus Cowan strain I, bacterial DNA and dsRNA,20-24 and Pertussis toxin in the context of IL-1
Incomplete knowledge exists regarding tissue factors that are encountered by DCs on their way to antigen presentation.11 Such factors may be decisively involved in fine-tuning DC polarization. An example for a Th1-inducing tissue factor is interferon-
Media and reagents RPMI 1640 was supplemented (complete RPMI [c-RPMI]) with 10% heat-inactivated fetal calf serum (FCS), 1 mM nonessential amino acids, 45 µg/mL penicillin and streptomycin, and 2 mM L-glutamine (all from Gibco, Eggenstein, Germany). For Th polarization assays, c-RPMI was supplemented with 5 x 10–5 M mercaptoethanol (Sigma-Aldrich, Taufkirchen, Germany). Recombinant fully mature human OPN was obtained from Chemicon (Hofheim, Germany).
Monoclonal antibodies (mAbs) specific for human leukocyte antigen DR (HLA-DR) (G46-6 (L243)), CD1a (HI149), CD80 (BB1), and CD86 (2331) were purchased from BD-Pharmingen (Heidelberg, Germany); and mAb for CD14 (RMO52), CD40 (MAB89), and CD54 (84H10), from Immunotech (Marseille, France). The anti–human integrin antibodies Split thickness skin organ culture The 1 cm x 2 cm split thickness skin (2-4 mm) was prepared by dermatome (Aesculap, Tuttlingen, Germany)30 and floated on c-RPMI in 6-well plates (Greiner, Frickenhausen, Germany) in the presence or absence of OPN (0.5 µg/mL). After 24 hours, cells that had migrated into the culture medium were collected and counted microscopically after lysis of erythrocytes. Migrated cells were stained with fluorescein isothiocyanate (FITC)–conjugated mAbs against CD1a, and for double staining additionally with phycoerythrin (PE)–conjugated anti–HLA-DR mAb or appropriate isotype controls, and were analyzed by fluorescence-activated cell sorter (FACS). The percentage of CD1a+ cells that migrate from split thickness skin, predominantly LCs, was calculated by CellQuest software (BD-Pharmingen). The number of LCs that had migrated was calculated by the following formula: LCs = absolute cell number (counted microscopically) x percentage of CD1a+ emigrated cells. Skin specimens were obtained from patients with elective plastic surgery, following informed consent. Approval for this study was obtained from the institutional review board of the University of Freiburg. Generation of human myeloid DCs Monocyte-derived DCs were prepared as described.31 In brief, CD14+ cells from peripheral blood mononuclear cells (PBMCs) were enriched with a bead-labeled anti-CD14 mAb (Miltenyi Biotec, Bergisch-Gladbach, Germany) using the MACS magnetic cell sorting system (Miltenyi Biotec). CD14+ cells (mean purity, 89.6%; SD ± 9.14%) (1 x 106/mL) were cultured for 5 days in C-RPMI containing granulocyte-macrophage colony-stimulating factor (GM-CSF, 1000 U/mL Leucomax; Novartis, Basel, Switzerland) and IL-4 (1000 U/mL; Promocell, Heidelberg, Germany) in 24-well culture plates to generate DCs. Cells were identified as immature DCs by positive expression of CD1a and the lack of CD14 (purity, 84.21%; SD ± 8.5%) and low expression of HLA-DR, CD40, CD80, and CD86.14 DC maturation/stimulation To study DC activation by OPN, immature DCs were harvested at day 5, extensively washed, and cultured (1 x 106) for 48 hours in c-RPMI in the presence or absence of 10 µg/mL LPS (Serotype O111:B4; Sigma-Aldrich), or 0.5 µg/mL OPN (Chemicon). Supernatants and cells were obtained after 24 and 48 hours. The chosen concentration was optimized in titration experiments for DC migration10 and stimulation of HLA-DR, CD40, and CD54 expression (Table 1).
Adhesion slides and immunostaining Stimulated DCs were adhered to BioRad adhesion slides (BioRad, Munich, Germany) according to the manufacturer's protocol. The slides were incubated with the primary anti–HLA-DR antibody followed by a biotinylated antimouse mAb and streptavidin peroxidase, both from LSAB-Kit (DAKO, Hamburg, Germany). The peroxidase was developed by 3-amino-9-ethyl carbazole according to the manufacturer's protocol. Flow cytometry Surface receptor expression on DCs was determined on days 5, 6, and 7. Cells were stained (4°C, 30 minutes) with FITC- or PE-labeled mAb. To exclude dead cells by appropriate gating, 2.5 µg/mL 7-aminoactinomycin D (7-AAD; Sigma-Aldrich) was added. Viable cells (5-10 x 103 per sample) were analyzed and mean fluorescence intensities (MFIs) determined by CellQuest software. Generation of human T cells PBMCs were isolated from blood of healthy donors by Ficoll gradient centrifugation.32 T cells for mixed lymphocyte reactions (MLRs) were enriched (purity, > 95%) by immunomagnetic negative-depletion using pan T-cell separation Kit and MACS columns (Miltenyi Biotec) according to the manufacturer's instructions. CD4+ cells for Th-cell polarization assays were isolated with the CD4 isolation Kit (Miltenyi Biotec) from PBMCs. For separation of CD45RA+ cells, CD4+ cells were incubated with PE-labeled anti-CD45R0 mAb, followed by incubation with anti-PE beads (Miltenyi Biotec) and separated by MACS. Mixed lymphocyte reaction (MLR) MLRs were performed at 2 different time points of DC culture. In setting one, day-5 immature DCs were used and OPN was present or absent throughout the MLR. In setting 2, day-5 DCs were left untreated for control, or stimulated with 0.5 µg/mL OPN or with 10 µg/mL LPS for 48 hours and then used for MLR. In both settings, MLRs were performed with a DC/T-cell (Tc) ratio of 1 x 104:1 x 105/200 µL in U-bottom 96-well plates (Greiner) using allogeneic pan T cells. For both MLR settings, allogeneic DCs and T cells were combined from the same donor pair. At both time points, maximum T-cell proliferation was induced by addition of phytohemagglutinin (PHA, 0.5 µg/mL) as positive control.
Supernatants were obtained after 48 hours of MLR to determine IL-12p70 and IL-10 secreted from DCs or IFN- Th-cell polarization assay
Naive CD45RA+ Th cells (20 x 103/200 µL) were enriched by immunomagnetic negative selection. They were then cocultured with DCs (5 x 103/200 µL) that had been prematured in the presence or absence of OPN (0.5µg/mL) or IFN- Cytokine measurement
Supernatants from DCs, MLR, and Th-cell polarization assays were harvested at the indicated time points and stored at –80°C. Cytokines were quantified by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions and measured at an extinction of 450 nm (MR5000 ELISA-reader and Bio-Linx Software; Dynatech, Chantilly, VA). The following ELISA kits were used (sensitivity): OPN (5 ng/mL) from IBL (Takasaki, Japan); IL-4 (7.8 pg/mL), IL-5 (7.8 pg/mL), IL-6 (4.7 pg/mL), IL-10 (7.8 pg/mL), IL-13 (2.1 pg/mL), IFN-
OPN activates human Langerhans cells, inducing their emigration from the epidermis and their phenotypical maturation Recently, we have demonstrated that OPN induces LC migration in vitro and in vivo in a murine CHS model.10 Whether OPN promotes LC/DC activation remains unclear. To explore whether OPN influences emigration of human LCs, thereby inducing their activation, we used a skin explant model in which LCs migrate from split thickness skin floated on culture medium with or without recombinant OPN.33,34 We found that the addition of OPN strongly induces the emigration of CD1a+ LCs from the epidermis (Figure 1A). LCs that had migrated upon OPN stimulation in skin explant cultures consistently showed up-regulated MHC II molecules compared with cells that had migrated into medium that was not OPN supplemented (Figure 1B). Our findings indicate that OPN promotes LC emigration from human skin and simultaneously induces signals promoting their maturation.
OPN induces DC differentiation
DCs have been shown to express OPN mRNA.35,36 We therefore investigated the OPN production of DCs generated from peripheral blood CD14+ monocytes. During their GM-CSF– and IL-4–driven differentiation from CD14+ monocytes into immature DCs on day 5, cells secreted 460.3 ng/mL (SD ± 214.2 ng/mL; n = 4, 1 x 106 cells/mL) OPN. To quantify the OPN secretion of the immature DCs after day 5, cells were extensively washed and recultured in fresh medium without cytokines. Within 24 hours, immature DCs secreted 313 ng/mL (SD ± 208.1 ng/mL; n = 4, 1 x 106 cells/mL) OPN. To investigate the effects of recombinant OPN on phenotypical myeloid DC maturation, washed DCs were stimulated with 0.5 µg/mL OPN. DCs were analyzed by light microscopy and flow cytometry at different time points of in vitro maturation.31 Immature DCs were either cultured without stimulus (Figure 2A-C) or treated with OPN (Figure 2D-F) or LPS as an established factor that initiates terminal DC maturation (Figure 2G-I) from culture days 5 to 7. In Figure 2, images were taken on days 6 (A,D,G) and 7 (B-C,E-F,H-I) of culture. When analyzing DC cultures by light microscopy, it was remarkable that OPN induced the formation of DC clusters (Figure 2). In contrast to unstimulated cultures (Figure 2A-B), addition of OPN induced DC clustering beginning 24 hours after stimulation (Figure 2D), which was further enhanced after 48 hours (Figure 2E). DCs cultured in the presence of OPN developed the characteristic dendrites of activated DCs (Figure 2F). Interestingly, they did not display the veils of DCs terminally matured by LPS treatment (Figure 2I).37 FACS analyses on days 5, 6, and 7 revealed a strong expression of CD1a, while CD14 was not detectable (Figure 3B), classifying these cells as DCs.31 In contrast to unstimulated cells, OPN treatment induced the expression of HLA-DR, CD40, CD80, CD86, CD44, and CD54 within 48 hours, in a manner comparable with that observed with LPS (Figure 3A; Table 1). Viability of DCs in the absence of OPN was 90.5% (SD ± 4.8%) on day 6 and 91.8% (SD ± 6.3%) on day 7. Addition of OPN did not affect DC viability (91% ± 3.7% on day 6, n = 6; 92.3% ± 5.5% on day 7, n = 4). DCs express the OPN receptors CD44 and the integrins
OPN stimulates the secretion of TNF- and IL-12 by DCsIn different systems, OPN has been shown to be central for the induction of an adequate Th1-type response.3,6,8,9,38 Because DCs play the key role for shaping T-cell–mediated immune responses through their secretion of specific cytokine patterns, we went on to determine the effect of recombinant OPN on the cytokine release of DCs.
Day-5 immature DCs (1 x 106/mL) were cultured in the presence or absence of OPN and supernatants were tested after 24 hours and 48 hours. Upon OPN stimulation, DCs secreted high amounts of TNF-
OPN enhances the allostimulatory capacity of DCs Having demonstrated that OPN induces DC activation, we went on to show that such activation is functional for the allostimulatory capacity of DCs. We had previously shown that OPN is up-regulated in the paracortical T-cell regions of skin-draining lymph nodes during the sensitization phase of CHS when DCs encounter T cells for activation.10 We now simulated this in vivo situation by adding recombinant OPN directly to DCs interacting with T cells in MLR. To address this, OPN was first added directly into the MLR with immature day-5 DCs. In this setting, OPN in contrast to control significantly augmented allogeneic T-cell proliferation (Figure 5A). Interestingly, if DCs were prestimulated by OPN from days 5 to 7 of culture, an even more pronounced increase in T-cell proliferation was detected (Figure 5B). These results were confirmed when the percentage of OPN-induced T-cell proliferation was calculated for each individual experiment and statistical comparison with untreated day-5 DCs (Figure 5C) or more mature day-7 DCs (Figure 5D) was performed. Comparing T-cell proliferation induced by day-5 DCs (open diamonds), addition of OPN to MLR significantly increased their potential to stimulate T-cell proliferation (Figure 5C open circles, mean: 1.33-fold, median: 1.25-fold). Percent induction of T-cell proliferation was even higher when DCs were OPN pretreated for 48 hours from days 5 to 7 of culture prior to MLR (Figure 5C closed circles, mean: 4.06-fold, median: 3.13-fold increase in T-cell proliferation). Although DCs cultured until day 7 without additional stimulus are more potent inducers of T-cell proliferation than immature day-5 DCs, as would be expected (Figure 5B), OPN treatment for the last 48 hours of culture further significantly enhanced their T-cell stimulatory capacity (Figure 5D closed circles; mean: 1.55-fold, median: 1.39-fold increase in T-cell proliferation). Similar results were obtained by direct addition of LPS to MLR or addition of LPS-prestimulated effector DCs (data not shown). Together with our findings that OPN induces changes in morphologic phenotype, cell surface protein expression, and cytokine secretion, these data suggest that OPN is a potent factor inducing functional DC activation.
In the presence of allogeneic T cells, OPN augments the production of IL-12p70 by DCs and of IFN-
In addition to the capacity of OPN-treated DCs to induce T-cell proliferation, we next analyzed whether Th cells were also polarized in their pattern of cytokine expression. IL-12 and IL-10 were measured to evaluate Th-polarizing cytokines secreted by the DCs and IFN-
Interestingly, when DCs were exposed to OPN or LPS for 48 hours prior to adding them to the MLR (Figure 6B,D), IL-10 secretion was reduced and only minimal amounts of IL-12 remained detectable (Figure 6B). When adding such DCs to MLR, OPN-prestimulated cells are still able to induce an increase of IFN- secretion but have lost their maximal polarizing capacity (Figure 6D). These findings provide strong evidence that OPN polarizes DC maturation toward a DC1 phenotype that enhances Th1 polarization by augmenting DC secretion of IL-12,18 thereby initiating Th1-cell induction. Furthermore, OPN-stimulated DCs only transiently produce IL-12 upon OPN encounter and become refractory to further T-cell–mediated stimulation.
OPN-activated DCs polarize naive T cells toward a Th1-, IFN-
It is the unique ability of DCs to induce a primary immune response through activation and polarization of naive T cells. Therefore, we determined the ability of OPN-activated DCs to induce a Th1 phenotype in naive (CD4+CD45RA+) Th cells. Enriched naive Th cells were cocultured with DCs that had been prestimulated for 12 hours prior to MLR in the presence of the superantigen SEB for 9 days. Experiments were performed either with allogeneic (Figure 7A) or autologous (Figure 7B) T cells. When cytokine secretion was induced by addition of PMA and ionomycin, we found that naive Th cells secreted substantially elevated amounts of the Th1 cytokine IFN-
Although OPN has been shown to have Th1 cytokine effects on T cells and macrophages in different in vivo and in vitro systems, it remained unknown whether OPN could exert its Th1-polarizing effects by influencing DCs. Here we demonstrate that OPN induces DC migration and activation, and polarizes them into Th1-promoting effector DCs. Our findings are important since they demonstrate that OPN as an endogenous environmental signal shapes the outcome of the immune response by DCs conditioned in peripheral tissues and during their encounter as APCs with naive T cells in secondary lymphatic tissues.
We have shown that murine DCs are guided to lymphatic tissues by OPN.10 Here we confirm that OPN induces the migration and maturation of human LCs as demonstrated in explant cultures of keratome skin. Human DCs express the OPN receptors CD44, v 3, v5, and 1 integrin. Interestingly, OPN, like LPS, induces the expression of the standard form of CD44 (CD44s), while the constitutive integrin expression was not further augmented. The OPN-induced CD44 expression by DCs may positively affect their migration to lymphatic tissues; as we have previously demonstrated, certain CD44 isoforms play a central role for LCs/DCs to migrate to and adhere in T-cell zones of lymph nodes.34,40 When further characterizing the OPN-induced DC phenotype, we found that OPN and LPS had a comparable potential to up-regulate MHC class II, costimulatory, and adhesion molecules (Figure 3). Importantly, although potently activating DC expression of these molecules, our morphologic observations (Figure 2) indicate that OPN induces a phenotype that probably represents a preterminal state of the LPS-driven myeloid-DC maturation pathway. However, we cannot exclude that OPN activation of the same CD14–CD1a+ immature DCs may induce a different pathway of DC maturation. Thus, OPN alone or in the context of other endogenous and exogenous factors enhances a distinct type of DC activation with T-cell stimulatory function. Recently Kawamura et al investigated the autocrine effects of OPN produced by DCs, neutralizing OPN with OPN-specific antibodies.41 OPN-deprived DCs had a reduced viability and expression of MHC class II and costimulatory molecules, which is in accordance to our findings that recombinant OPN induces DC activation (Figure 3A). Comparable with this work, we found that OPN is secreted during the GM-CSF– and IL-4–driven differentiation of CD14+ monocytes into DCs. Immature DCs washed and cultured in the absence of GM-CSF and IL-4 secreted OPN in a range of 300 ng/mL within 24 hours. In our system, following elimination of endogenous OPN, the addition of recombinant OPN induced DC maturation that was more pronounced than that of washed DCs cultured in the absence of OPN containing DC-conditioned medium (Figure 3C). Most likely in this setting the immediate increase in OPN concentration initiates the activation signal. By using different systems, both studies indicate that OPN is an important autocrine maturation stimulus for DCs. Furthermore, we found that additional exogenous OPN in the presence of self-produced OPN (Figure 3C) induces DC activation. This may be explained by an additive effect. However, it cannot be excluded that the recombinant OPN, due to missing posttranslational modifications, has different receptor avidity.
IL-12 produced by DCs is the critical Th1-polarizing cytokine for CD4+ T cells and stimulates the secretion of IFN- Inspired by the concept that the IL-12–producing capacity of DCs, which migrate into the regional lymphatic tissue, is preestablished in the tissue of their origin and instructed by tissue environmental factors,28,49 we investigated whether OPN influences the Th-polarizing capacity of DCs. Indeed OPN enhanced the Th1-driving phenotype of DCs, because we found that OPN induced the secretion of IL-12p70 to amounts within the range of 0.03 to 1.0 ng/mL, a concentration that has been described by Snijders et al to direct the development of naive Th cells into Th1 cells.20 In our experiments, the level of IL-12p70 showed some variation among different donors. This may be explained by donor variability, for example a Th2-primed state of DCs. Although for all experiments only DCs from healthy donors were used, one cannot exclude a latent atopic disposition of some donors, which is accompanied by a Th2-polarized immune response.25
CD40 ligand (CD40L) and IFN-
In contrast to IFN-
In peritoneal macrophages, OPN engagement with In several disease models with OPN-deficient mice the role of OPN as a Th1-driving cytokine has been established.6,61-64 OPN-deficient mice show a milder course and improved clinical outcome of an experimental autoimmune encephalitis.8,9 These studies compared Th-polarizing cytokines from OPN-deficient or wild-type mice in mixed cell populations generated from regional lymph nodes or spleen. In such cell populations, immune cells from OPN-deficient mice secreted fewer Th1-driving cytokines, while increased levels of antagonistic Th2 cytokines (eg, IL-10) were detected. In the study by Ashkar et al, OPN was shown to differentially regulate IL-12 secretion by peritoneal macrophages.6 However, although macrophages shape the type of immune response at the site of inflammation, they are not the primary cells to leave such sites to modulate the immune response in secondary lymphatic organs.65 In contrast to macrophages, DCs are able to encounter the antigen at the site of inflammation and thereafter invade regional secondary lympoid organs to initiate antigen-specific cell-mediated immune response. Here we show that OPN early during DC activation induces polarization of DCs toward a Th1-inducing phenotype. We therefore propose that OPN-polarized DCs may be of central importance for the initiation of the Th1-modulated cytokine milieu detected in lymph nodes of the mentioned disease models. In conclusion, our findings suggest that OPN is a tissue factor expressed during inflammatory responses that, through its ability to activate DCs, in concert with other cytokines, shapes T-cell–mediated immune responses. Furthermore, OPN may decisively influence T-cell polarization by its presence when DC–T-cell interaction occurs after DCs have entered lymph nodes. This concept is underlined by the deficiency of Th1 immunity in OPN-deficient mice, as in these mice the absence of OPN may lead to a lack in priming of DCs toward the Th1-initiating phenotype.
The authors thank Christine Bernardi for excellent technical assistance.
Submitted August 25, 2004; accepted March 23, 2005.
Prepublished online as Blood First Edition Paper, April 26, 2005; DOI 10.1182/blood-2004-08-3228.
Supported by grants from the Deutsche Forschungsgemeinschaft (DFG WE 1919/2-3 and SFB 620, TP B5).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Andreas C. Renkl, University of Ulm, Department of Dermatology and Allergology, Maienweg 12, 89081 Ulm, Germany; e-mail: andreas.renkl{at}medizin.uni-ulm.de.
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Abstract
Introduction
) were cultured for 24 hours in 100% (
) or 50% (
) DC-conditioned medium or in fresh medium without GM-CSF or IL-4 (0%; 
), with (+) or without (–) exogenous OPN. Corrected mean fluorescence (cMFI) of HLA-DR expression from 1 representative of 3 independent experiments is given.
). Alternatively, DCs were prestimulated with OPN for 48 hours prior to addition to MLR (
). The allostimulatory capacity in both settings was compared with that of immature DCs from day 5 (open gray diamonds). For statistical analysis of all experiments, the percent induction of T-cell proliferation by OPN was calculated by the following formula: T-cell proliferation induced by prestimulated DCs or DCs in the presence of OPN (CPM)/T-cell proliferation induced by immature DCs of day 5 (CPM) x 100%. Statistical analysis of 7 independent experiments ± SEM (*P < .05, one-way analysis of variance [ANOVA], Student-Neuman-Keuls test). (D) The stimulatory capacity of 48-hour OPN-stimulated DCs (
B (NF-