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Institute of Immunology, Center of Pathophysiology, Infectiology, and Immunology, Medical University of Vienna, Lazarettgasse 19, Vienna A-1090, Austria.
Institute of Immunology, Center of Pathophysiology, Infectiology, and Immunology, Medical University Vienna, Vienna, AustriaInstitute of Pathophysiology and Immunology, Center of Molecular Medicine, Medical University Graz, Graz, Austria
The epithelial signaling protein and transcriptional regulator β-catenin has recently been implicated in hematopoietic dendritic cell (DC) differentiation as well as in DC-mediated tolerance. We here observed that epidermal Langerhans cells (LCs) but not interstitial/dermal DCs express detectable β-catenin. LCs are unique among the DC family members in that LC networks critically depend on epithelial adhesion molecules as well as on the cytokine transforming growth factor-β1 (TGF-β1). However, despite the important functions of LCs in the immune system, the molecular mechanisms governing LC differentiation and maintenance remain poorly defined. We found that TGF-β1 induces β-catenin in progenitor cells undergoing LC differentiation and that β-catenin promotes LC differentiation. Vitamin D, another epidermal signal, enhanced TGF-β1-mediated β-catenin induction and promoted the expression of multiple epithelial genes by LCs. Moreover, full-length vitamin D receptor (VDR) promoted, whereas a truncated VDR diminished, the positive effects of ectopic β-catenin on LC differentiation. Therefore, we here identified β-catenin as a positive regulator of LC differentiation in response to TGF-β1 and identified a functional interaction between β-catenin and VDR in these cells.
Abbreviations
CHX
cyclohexamide
DC
dendritic cell
GSK3β
glycogen synthase kinase 3β
HPC
hematopoietic progenitor cell
LC
Langerhans cell
qRT–PCR
quantitative real-time reverse-transcriptase–PCR
TGF-β1
transforming growth factor-β1
VDR
vitamin D receptor
1,25VD3
1α,25-dihydroxyvitamin D3
Introduction
Langerhans cells (LCs) represent abundantly occurring and evolutionarily highly conserved dendritic cells (DCs) specifically located in the stratified epithelial tissues. LCs are unique among DC family members in that they express epithelial-type adhesion molecules, allowing them to form a tight three-dimensional network in the basal and suprabasal epidermal keratinocyte layers. LCs are potent inducers of adaptive immune responses and possess innate immune functions (
). However, the epithelial microenvironmental mechanisms underlying LC development, adhesion, and function are still poorly defined.
β-Catenin is a transcriptional regulator of the Wnt signaling pathway. It interacts with the intracellular tail of cadherins such as E-cadherin and thereby provides a link of cadherins to the cytoskeleton (
). We previously demonstrated that E-cadherin is induced by transforming growth factor-β1 (TGF-β1) during LC differentiation from CD34+ human hematopoietic progenitor cells (HPCs) (
). A hallmark of in vitro generated LCs is their tendency to form large, macroscopically visible homotypic cell clusters. These LC clusters are at least partially mediated through E-cadherin adhesion (
). Moreover, we showed that in response to mechanical cluster disaggregation, LCs undergo activation/maturation and specific E-cadherin engagement immediately after cluster disruption inhibited their maturation (
). Murine studies subsequently demonstrated that this effect is mediated by β-catenin signaling due to the loss of E-cadherin adhesion and that cluster-disrupted DCs exert a tolerogenic function upon transfer in vivo (
). Recently, it was shown that intestinal lamina propria DCs and macrophages express higher levels of transcriptionally active β-catenin as compared with splenic DCs, and the deletion of β-catenin from CD11c+ cells promoted the occurrence of intestinal autoimmunity (
), data on the expression distribution of β-catenin in DC subsets are lacking. Nevertheless, a role of β-catenin in DC subset differentiation has been demonstrated. One study showed that DC and monocyte differentiation was inhibited by β-catenin agonistic stimuli, Wnt3a and LiCl (
LCs and interstitial-type/dermal DCs can be generated from human CD34+ HPCs in response to specific cytokine combinations. In this differentiation model, interstitial/dermal DCs develop from monocyte intermediates in the absence of TGF-β1. Conversely, the addition of TGF-β1 early after culture onset redirects monocyte to LC differentiation. Data on β-catenin expression and function in cells undergoing LC versus monocyte differentiation are lacking. As LCs represent a unique DC subset that has recently been shown to negatively regulate immune responses (
), we here studied the role of β-catenin in these cells in vitro.
Results
TGF-β1 induces β-catenin expression during LC subset differentiation
We analyzed β-catenin expression using human skin-resident DCs. LCs identified as HLA-DR+ epidermal cells express similar high β-catenin levels as observed for keratinocytes. Conversely, dermal HLA-DR+ cells (dermal DCs and macrophages) exhibit low to undetectable β-catenin expression (Figure 1a). LCs can be generated in vitro from human CD34+ HPCs in response to TGF-β1 stimulation (
). Specifically, the addition of TGF-β1 to a basic cytokine combination comprising GM-CSF, stem cell factor, fms-related tyrosine kinase 3 ligand, and tumor necrosis factor-α in defined serum-free medium results in LC differentiation from CD34+ HPCs. Omission of TGF-β1 from these cultures abrogates LC differentiation in favor of CD14+CD11b+ monocytes. Therefore, in this model TGF-β1 induces LCs at the expense of monocyte differentiation. It is to be noted that percentages of LCs drop substantially upon omission of TGF-β1 from cultures between day 4 and day 7, indicating that in vitro LC differentiation is critically dependent on the continuous presence of TGF-β1 (Supplementary Figure S1a, b online). Quantitative real-time reverse-transcriptase–PCR (qRT–PCR) analysis revealed that β-catenin is expressed at substantially higher levels by day 7–generated cells from TGF-β1-supplemented LC generation cultures as compared with TGF-β1-nonsupplemented monocyte generation cultures (Figure 1b).
Figure 1Transforming growth factor-β1 (TGF-β1) induces β-catenin expression during Langerhans cell (LC) differentiation. (a) Immunofluorescence staining of human skin sections for HLADR (red) and β-catenin (green). Nuclei were visualized with 4',6-diamidino-2-phenylindole (DAPI). Scale bar=10μm, n=3. (b) Quantitative real-time reverse-transcriptase–PCR (qRT–PCR) analysis of β-catenin expression in LC (+TGF-β1) or monocyte (-TGF-β1) generation cultures at day 7 (d7). Values were normalized to HPRT (n=4, ±SD, *P<0.05). (c) CD34+ hematopoietic progenitor cells (HPCs) were pretreated with protein synthesis inhibitor cyclohexamide (CHX) at a concentration of 0.05ngml−1 for 1hour before addition of TGF-β1 (0.5ngml−1). RNA samples were collected at 0 and 6hours. mRNA of β-catenin was quantified by qRT–PCR (n=4, ±SD). (d) Cells analyzed for β-catenin, CD207, and CD14 expression. (e) β-Catenin immunoblot analysis of day 7–generated cells compared with CD34+ cells.
Because TGF-β1 instructs LC differentiation from HPCs, we studied whether β-catenin is induced in progenitor cells at early time points upon TGF-β1 addition. This was indeed the case (Figure 1c). Moreover, TGF-β1-mediated β-catenin induction did not require new protein synthesis as revealed from cyclohexamide (CHX) addition experiments (Figure 1c). Flow cytometry and immunoblot analyses confirmed that TGF-β1-induced CD207+CD1a+ LCs expressed higher levels of β-catenin compared with CD207−CD1a− cells from the same cultures. Moreover, the majority of CD14+ or CD14− cells from TGF-β1-nonsupplemented monocyte generation cultures lacked detectable β-catenin expression (Figure 1d and e). Therefore, LC differentiation is marked by the induction of high levels of β-catenin, congruent with the observation that among skin monocyte/DC subsets only LCs expressed substantial β-catenin levels.
β-Catenin promotes LC differentiation
We analyzed whether β-catenin promotes LC differentiation. FACS and western blot analyses of gene-transduced cells confirmed that the forced retroviral expression or lentiviral short hairpin RNA–mediated knockdown of β-catenin modulated β-catenin levels (Supplementary Figure S2a–d online). Ectopic β-catenin augmented the generation of CD1a+CD207+ LCs and this effect required TGF-β1. In the absence of TGF-β1, β-catenin instead promoted the generation of CD1a+ cells lacking CD207+ (Figure 2a). In contrast, knockdown of β-catenin during LC generation resulted in reduced percentages of LCs (Figure 2b).
Figure 2β-Catenin promotes Langerhans cell (LC) differentiation. (a) CD34+ hematopoietic progenitor cells (HPCs) were transduced with retroviral vector encoding β-catenin-IRES-GFP or empty control vector and cultured in LC/monocyte generation conditions (±transforming growth factor-β1 (TGF-β1)). Day 7 GFP+ cells were analyzed for CD1a and CD207. (b) CD34+ HPCs were transduced with lentiviral short hairpin RNA (shRNA)-β-catenin-pLKO1-eGFP and cultured in LC generation conditions (+TGF-β1). Day 7 GFP+ cells were analyzed for CD1a and CD207. (c) Schematic representation of experimental set up. LC or monocyte (Mo) generation cultures (±TGF-β1) were initiated in the presence or absence of glycogen synthase kinase 3β (GSK3β) inhibitor±SB216763 (1nMml−1, n=3). (d) FACS analysis of day 7–generated cells (±SB216763). Bar diagrams represent percentages and total numbers of CD1a+CD207+ cells (n=4). (e) Cell morphology of LC generation cultures (±SB216763). (f) Cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) at day 0 and analyzed at day 2 (n=4). *P=0.01; **P=0.001.
GSK3β is known to phosphorylate β-catenin at serine and threonine residues in the NH2-terminal region, which results in proteasome-mediated degradation of β-catenin (
). We added the GSK3β inhibitor SB216763 or LiCl, another β-catenin agonist, to LC or monocyte differentiation cultures as schematically shown in Figure 2c and Supplementary Figure S3a online. As expected, both compounds increased β-catenin protein levels in differentiated LCs (Supplementary Figures S2e and f and S3b online). SB216763 or LiCl promoted LC differentiation in the presence of TGF-β1 (Figure 2d and Supplementary Figure S3c online). This effect was accompanied by increased numbers of macroscopically visible LC clusters (Figure 2e and Supplementary Figure S3d online). In addition, elevated β-catenin levels failed to modulate cell proliferation as evidenced from carboxyfluorescein succinimidyl ester labeling experiments (Figure 2f and Supplementary Figure S3e online). Together, these results demonstrate that enforced β-catenin expression in HPCs promotes their differentiation into LCs.
TGF-β1 induces multiple epithelial genes during LC differentiation
β-Catenin is a key regulator of adherens junctions and cell differentiation. Consistently, β-catenin activation enhanced LC cluster formation (Figure 2e and Supplementary Figure S3d online), an effect at least partially dependent on E-cadherin adhesion (
). In addition to E-cadherin, LCs express other epithelial adhesion–associated molecules such as claudin-1, epithelial cell adhesion molecule (EpCAM), and TROP2 (
). We analyzed whether TGF-β1-dependent LCs express multiple epithelial adhesion molecules. TGF-β1-induced LCs express cytokeratin-18 (CK18), cytokeratin-8 (CK8), ZO3, JAM-1, and occludin (Ocln) at mRNA levels (Figure 3b). Conversely, cells from monocyte cultures (-TGF-β1) showed low to undetectable epithelial gene expression levels (Figure 3b). Flow cytometry analysis confirmed these data (Figure 3c). Pretreatment of CD34+ HPCs with CHX before addition of TGF-β1 inhibited the induction of epithelial genes within 6hours (Figure 3d), indicating that the observed epithelial gene induction in LCs by TGF-β1 requires new protein synthesis. In conclusion, LC differentiation in response to TGF-β1 is accompanied by the induction of multiple epithelial genes.
Figure 3Transforming growth factor-β1 (TGF-β1) induces epithelial gene expression during Langerhans cell (LC) differentiation. (a) Schematic representation of in vitro generated cells. (b) Quantitative real-time reverse-transcriptase–PCR (qRT–PCR) expression analysis of epithelial genes in LC (+TGF-β1) or monocyte (Mo) (-TGF-β1) generation cultures. Values were normalized to HPRT (±SD, n=7, *P<0.05). (c) Overlay histograms represent mean fluorescence intensities (MFIs) of epithelial genes expressed by gated CD1a+CD207+ cells (black lines) at day 7. Total cells from cultures without TGF-β1 are compared (solid gray). FACS diagrams represent cells from TGF-β1-dependent LC generation cultures analyzed for epithelial gene expression. (d) CD34+ hematopoietic progenitor cells (HPCs) were pretreated with protein synthesis inhibitor cyclohexamide (CHX) as described in Figure 1c. RNA samples were collected at 0 and 6hours. mRNA of epithelial genes was quantified using qRT–PCR. Error bars represent the mean of four independent experiments (±SD). **P=0.001.
). Moreover, it is known that epithelial adhesion molecules are positively regulated by VD3/VDR signaling because of the presence of vitamin D response elements (
). Therefore, we studied the effects of 1α,25-dihydroxyvitamin D3 (1,25VD3) on LC differentiation and epithelial gene expression. The vitamin D metabolites 25VD3 and 1,25VD3 can be detected in human skin (
). Moreover, we have already described that the ectopic expression of the VDR promotes TGF-β1-dependent LC differentiation and an inhibitor of VDR/RXRα interfered with LC generation (
). We added 1,25VD3 to LC generation cultures from days 4 to 6 (Figure 4a). Percentages of CD1a+CD207+ cells were enhanced upon 1,25VD3 addition as compared with control (Figure 4b). Moreover, LCs generated in the presence of 1,25VD3 exhibited elevated expression levels of epithelial molecules (Figure 4c). Inversely, β-catenin inhibition during LC differentiation resulted in reduced expression of epithelial genes (Supplementary Figure S4 online). Therefore, 1,25VD3 promotes LC differentiation and this effect is associated with the enhancement of epithelial gene expression by LCs. In addition, 1,25VD3 increased the expression levels of VDR, TGF-β1, and β-catenin by LC precursors (Figure 4d), i.e., positive regulators of LC differentiation.
Figure 41α,25-Dihydroxyvitamin D3 (1,25VD3) promotes Langerhans cell (LC) differentiation along with epithelial gene expression. (a) Schematic representation of 1,25VD3 treatment of LCs. Mo, monocyte. (b) Analysis of day 7–generated cells. Bar diagrams represent percentages and total numbers of CD1a+CD207+ cells (n=3). NS, not significant. (c) Mean fluorescence intensities (MFIs) of epithelial genes expressed by LCs (+1,25VD3, gated CD1a+CD207+; n=3). (d) Quantitative real-time reverse-transcriptase–PCR (qRT–PCR) analysis of transforming growth factor-β1 (TGF-β1), vitamin D receptor (VDR), and β-catenin mRNA expression by 1,25VD3-treated LCs (n=3, ±SD, *P<0.05). (e) qRT–PCR analysis of VDR expression by day 7–generated LCs (n=4, ±SD, *P<0.05). (f) CD34+ hematopoietic progenitor cells (HPCs) were pretreated with cyclohexamide (CHX) as described in Figure 1c. VDR mRNA was quantified by qRT–PCR (n=4, ±SD). (g) CD34+ HPCs were transduced with a retroviral vector encoding VDR-IRES-GFP or empty control vector. Day 7 GFP+ cells were analyzed for CD1a and CD207. (h) Overlay histograms represent E-cadherin (E-cad) expression by gated GFP+CD1a+CD207+ cells (n=3). **P=0.008; ***P=0.001.
VDR promotes LC differentiation along with enhancement of E-cadherin expression
The day 7–generated LCs (+TGF-β1) expressed higher VDR levels compared with monocytes (-TGF-β1) (Figure 4e). TGF-β1-mediated VDR induction did not require new protein synthesis as revealed from CHX addition experiments. In fact, VDR levels were higher in CHX-treated cells compared with control, indicating that TGF-β1 may induce VDR together with a VDR repressor or another negative regulator of VDR (Figure 4f). Moreover, ectopic VDR promoted LC differentiation, in keeping with the above-described positive effects of VD3 on percentages of LCs (Figure 4b). Moreover, VDR-transduced LCs exhibited increased levels of E-cadherin expression compared with control-transduced cells (Figure 4g and h).
β-Catenin cooperates with VDR during LC differentiation
We next analyzed whether ectopic VDR and β-catenin show redundant or additional effects on LC generation. Combined transduction of progenitor cells with VDR and β-catenin resulted in the enhancement of LC differentiation relative to each alone, with very high percentages of LCs generated from double gene–transduced cells (Figure 5a). Moreover, the coexpression of VDR.deltaAF2 with β-catenin diminished the positive effect of β-catenin on LC differentiation (Figure 5a), indicating that β-catenin requires VDR for promoting LC differentiation from HPCs.
Figure 5Positive effects of β-catenin and VD3/vitamin D receptor (VDR) during Langerhans cell (LC) differentiation. (a) CD34+ hematopoietic progenitor cells (HPCs) were transduced with vectors encoding VDR-IRES-GFP, β-catenin-IRES-NGFR, or VDR-delta AF2-IRES-GFP. FACS plots represent day 7–generated GFP+ cells. Bar diagrams represent percentages of CD1a+CD207+ cells among GFP+ cells (n=3, *P<0.05). (b) LCs were treated with SB216763 and/or 1α,25-dihydroxyvitamin D3 (1,25VD3) as above (Figures 2c and 4a) and analyzed at day 7. Bar diagrams represent percentages and total numbers of CD1a+CD207+ cells (n=3, *P<0.05). NS, not significant. (c) Quantitative real-time reverse-transcriptase–PCR (qRT–PCR) analysis of T-cell factor (TCF) by day 7–generated LCs (n=3, ±SD, *P<0.05). (d) HEK293 cells cotransfected with a TCF/LEF reporter and expression plasmids. Luciferase values were normalized to β-galactosidase (β-gal; n=3). **P=0.001.
Accordingly, LC cultures supplemented with β-catenin activator SB216763 plus 1,25VD3 exhibited consistently increased percentages of LCs relative to cultures containing each compound alone (n=3, Figure 5b). Ectopic expression of VDR resulted in elevated expression levels of β-catenin (Supplementary Figure S5b online).In addition, the forced expression of β-catenin resulted in slight increase in the VDR expression (Supplementary Figure S5a online).
In subsequent experiments, we analyzed whether the β-catenin effect on LC differentiation might depend on T-cell factor (TCF) or VDR. If β-catenin forms a complex with VDR in epithelial cell lines, it does not activate TCF-binding sites (
). LCs expressed significant levels of TCF (Figure 5c) together with VDR (Figure 4e). We therefore asked whether the β-catenin effect on LC differentiation might depend on TCF or VDR. Cotransfection of HEK293 cells with TCF reporter along with β-catenin and VDR confirmed the previous observations (
) that β-catenin activated TCF reporter when expressed alone, whereas the coexpression of VDR plus β-catenin completely abrogated TCF reporter activity (Figure 5d). VDR/β-catenin expression levels in gene-transduced LCs and transfected HEK293 cells are likely similar. Although formal demonstration of this effect in primary LCs was technically not feasible, these data indicate that high VDR levels in LCs may compete with TCF for β-catenin binding. These observations suggest that LC differentiation is positively regulated by a similar VDR/β-catenin interaction–dependent mechanism previously described for epithelial cells.
VDR and β-catenin signaling promote the immunostimulatory capacity of LCs
A hallmark characteristic of LCs is their potent capacity to induce allogeneic T-cell proliferation. We therefore studied whether treatment of LCs with 1,25VD3 and SB216763 (a condition where β-catenin, VDR, and TGF-β1 are higher) promotes the immunostimulatory capacity of LCs. LCs generated in the absence or presence of these compounds were cocultured with allogeneic T cells. Cluster-purified LCs stimulated with 1,25VD3 plus SB216763 exhibited a higher T-cell proliferation capacity as compared with control cells (Supplementary Figure S6 online).
In conclusion, we demonstrated that TGF-β1 coinduces two transcription factors that enhance LC differentiation, i.e., β-catenin and VDR. According to our model, TGF-β1 and vitamin D3, the two epidermal microenvironmental signals, are critical for LC phenotype induction (Supplementary Figure S7 online).
Discussion
We here showed that among skin-resident monocyte/DC family members, high levels of β-catenin expression are confined to epidermal LCs and that TGF-β1 induces β-catenin in progenitor cells undergoing LC commitment. Our mechanistic data showed that β-catenin promotes LC differentiation from HPCs. Moreover, we found that TGF-β1 and β-catenin intersect with vitamin D signaling during LC differentiation. First, 1,25VD3 stimulation of LC precursors increased the expression levels of β-catenin along with TGF-β1 and VDR. Accordingly, 1,25VD3 promoted the TGF-β1-induced expression of multiple epithelial genes by LCs. Second, the positive effects of β-catenin on LC generation were diminished when a mutant VDR lacking the previously defined VDR/β-catenin physical interaction domain (i.e., VDR.deltaAF2) was coexpressed in HPCs. Therefore, VDR and β-catenin may represent critical positive regulators of LC differentiation.
VDR is downregulated during monocyte-derived DC differentiation (
). Therefore, among DC family members a possible cooperative effect of VDR and β-catenin might be specific for LC development and maintenance. In support of this assumption, VDR inhibition impaired LC generation in vitro (
). Here, we showed that LCs express several additional epithelial adhesion molecules, i.e., CK18, CK8, ZO3, JAM-1, and Ocln. Together, these data indicate that TGF-β1 induces an epithelial gene expression program during LC differentiation from HPCs.
We provided evidence for a role of VDR and β-catenin during LC differentiation downstream of TGF-β1 and VD3. Studies in epithelial cells previously identified a critical physical interaction between VDR and β-catenin during differentiation and epithelial gene expression. β-Catenin specifically activates VDR at the expense of TCF transcriptional activity depending on the availability of either factor (
). For example, during hair follicle differentiation, combined activation of VDR and β-catenin signaling promoted the formation of VDR/β-catenin complexes (
). The VDR.deltaAF2 mutant used in our study was previously used to demonstrate the formation of functional VDR/β-catenin complexes in epithelial cells (
). We here described that VDR.deltaAF2 diminishes the positive effects of β-catenin on LC differentiation, indicating that LC differentiation is positively regulated by the same VDR/β-catenin cooperation previously established in epithelial cells. This model is further supported by the findings that 1,25VD3 promoted epithelial gene expression of LCs and that the ectopic coexpression of VDR plus β-catenin resulted in substantially elevated percentages of generated LCs relative to each factor alone. Lentiviral short hairpin RNA knockdown resulted in reduced β-catenin expression levels, and this effect correlated with reduced percentages of generated LCs. Importantly, β-catenin was only partially inhibited in these experiments. Whether β-catenin is strictly required for LC differentiation remains to be studied.
Our observation that TGF-β1 induces β-catenin in HPCs undergoing LC commitment is reminiscent of the demonstration that nuclear translocation of β-catenin in bone marrow–derived mesenchymal stem cells occurs through the activation of the TGF-β1 signaling pathway (
Smad3-dependent nuclear translocation of beta-catenin is required for TGF-beta1-induced proliferation of bone marrow-derived adult human mesenchymal stem cells.
). In contrast, our observations that ectopic VDR promotes TGF-β1-dependent LC differentiation in the absence of vitamin D supplementation might be related to the findings that the in vitro binding of VDR and Smad3 is ligand independent (
Our data indicate that the two microenvironmental signals VD3 and TGF-β1 intersect for inducing LC differentiation and associated epithelial gene expression. Interestingly, β-catenin (
) have previously been implicated in the generation of tolerogenic DCs, and 1,25VD3 stimulation of LCs enhance their capacity to induce regulatory T cells (
). Augmentation of LC phenotypic characteristics by these signals is in line with the recent demonstration that LCs exert a regulatory function in vivo (
). Enhancement of mixed leukocyte reaction activity by LCs upon VD3 plus GSK3β inhibitor treatment correlated with the augmentation of LC phenotypic characteristics. Therefore, these compounds might be added to LC generation cultures to obtain potent antigen-presenting LCs ex vivo for basic and clinically oriented studies. The observed enhanced allogeneic-mixed leukocyte reaction activity by VD3/β-catenin agonist–treated LC precursors is supportive of our phenotypic data that these signaling pathways promote LC differentiation from progenitor cells.
Inducible LC-specific abrogation of autocrine/paracrine TGF-β1 signaling led to the migration of epidermal LCs into skin-draining lymph nodes, with migrated LCs maintaining the phenotype of homeostatically migrated nonactivated LCs (
). Our study shows that β-catenin and E-cadherin are coinduced during TGF-β1-dependent LC differentiation, supporting that β-catenin represents an important signaling molecule during LC activation. However, we did not study LC activation/maturation; therefore, it will be interesting to study the roles of TGF-β1 and E-cadherin/β-catenin signaling during these processes using the in vitro model described here.
Materials and Methods
Cytokines and reagents
Human stem cell factor, thrombopoietin, tumor necrosis factor-α, GM-CSF, and fms-related tyrosine kinase 3 ligand were obtained from PeproTech (London, UK); TGF-β1 was purchased from R&D Systems (Wiesbaden, Germany). GSK3β inhibitor (SB216763), CHX (C-6255), and 1,25VD3 were purchased from Sigma-Aldrich (St Louis, MO).
Isolation and in vitro culture of CD34+ cord blood cells
CD34+ cells were isolated as previously described (
flt3 ligand in cooperation with transforming growth factor-beta1 potentiates in vitro development of Langerhans-type dendritic cells and allows single-cell dendritic cell cluster formation under serum-free conditions.
). Murine mAbs of the following specificities were used: FITC-conjugated mAb specific for CD14; biotinylated mAb specific for αNGFR, conjugated with streptavidin-PerCP (BD Biosciences, Palo Alto, CA); phycoerythrin-conjugated mAb specific for CD207 (Immunotech, Marseille, France); allophycocyanin-conjugated antibody specific for CD324 and Pacific Blue–conjugated CD1a (BioLegend, San Diego, CA). For intracellular staining, cells were fixed with fixation medium (An der Grub, Kaumberg, Austria) for 5minutes at room temperature and permeabilized with permeabilization medium (An der Grub). Later, cells were stained with mAbs specific for CK18, CK8 (Sigma-Aldrich), claudin-1 (Zymed Laboratories, Vienna, Austria), polyclonal antibodies specific for JAM-1, ZO3, and Ocln (Invitrogen, Vienna, Austria), or isotype controls (Cell Signaling Technologies, Beverly, MA). Second-step reagent was Alexa 647–labeled goat anti-rabbit IgG or rabbit anti-mouse IgG (Invitrogen). Flow cytometric analysis was performed using a LSRII instrument (BD Biosciences) and the FlowJo software (Tree Star, Ashland, OR).
Immunohistochemistry
Human tissue specimens were stained as described (
). For detection of β-catenin antibody, slides were probed with a polyclonal donkey-anti-mouse Alexa-Fluor-488-conjugated antibody (Invitrogen). The biotin-labeled HLA-DR antibody was detected using streptavidin-phycoerythrin (BD Biosciences). Nuclei were stained with 4',6-diamidino-2-phenylindole. Pictures were taken using an LSM700 microscope and Zen 2009 software (Carl Zeiss, Vienna, Austria).
Luciferase assay
Luciferase assay was performed as previously described (
). 293T cells were transfected with the TCF/LEF reporter luciferase construct, kindly provided by Dr Peter Petzelbauer (Department of Dermatology, Medical University of Vienna, Vienna, Austria) or empty control vector or the indicated expression plasmids.
T-cell proliferation assays
T-cell proliferation assay was performed as described before (
Statistical analysis was performed using the paired, two-tailed Student’s t-test or analysis of variance; P-values of <0.05 were considered significant.
ACKNOWLEDGMENTS
We thank W Ellmeier, Institute of Immunology, Medical University Vienna, for critically reading the manuscript and for helpful discussion. This study was supported by the Austrian Science Fund Research grants FWF-Lise-Meitner-Stipend M1096-B13 to SK; P22058, P19245, and SFB2304 to HS; PhD program W1212 “Inflammation and Immunity” to GE and TB; and a PhD fellowship of the High Education Commission of Pakistan to NY.
Author contributions
NY, SK, GE, YMS, MS, and TB performed experiments and analyzed data; JS analyzed data; NY and HS wrote the paper and designed the study; and HS supervised the work.
Smad3-dependent nuclear translocation of beta-catenin is required for TGF-beta1-induced proliferation of bone marrow-derived adult human mesenchymal stem cells.
flt3 ligand in cooperation with transforming growth factor-beta1 potentiates in vitro development of Langerhans-type dendritic cells and allows single-cell dendritic cell cluster formation under serum-free conditions.
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