The interplay between surfaces and soluble factors define the immunologic and angiogenic properties of myeloid dendritic cells
© Sprague et al; licensee BioMed Central Ltd. 2011
Received: 23 October 2010
Accepted: 6 June 2011
Published: 6 June 2011
Dendritic cells (DCs) are antigen presenting cells capable of inducing specific immune responses against microbial infections, transplant antigens, or tumors. Interestingly, microenvironment conditions such as those present in tumor settings might induce a DC phenotype that is poorly immunogenic and with the capability of promoting angiogenesis. We hypothesize that this plasticity may be caused not only by the action of specific cytokines or growth factors but also by the properties of the surfaces with which they interact, such as extracellular matrix (ECM) components.
Herewith we studied the effect of different surfaces and soluble factors on the biology of DCs. To accomplish this, we cultured murine myeloid(m) DCs on surfaces coated with fibronectin, collagen I, gelatin, and Matrigel using poly-D-lysine and polystyrene as non-biological surfaces. Further, we cultured these cells in the presence of regular DC medium (RPMI 10% FBS) or commercially available endothelial medium (EGM-2). We determined that mDCs could be kept in culture up to 3 weeks in these conditions, but only in the presence of GM-CSF. We were able to determine that long-term DC cultures produce an array of angiogenic factors, and that some of these cultures still retain the capability to induce T cell responses.
Altogether these data indicate that in order to design DC-based vaccines or treatments focused on changing the phenotype of DCs associated with diseases such as cancer or atherosclerosis, it becomes necessary to fully investigate the microenvironment in which these cells are present or will be delivered.
Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone marrow, spleen, lymph nodes and Peyer's patches [1–3]. In the mouse, DCs can be broadly divided into plasmacytoid and myeloid DCs . Plasmacytoid DCs (pDCs) are characterized by the expression of B220 but no CD11b and produce large amounts of type-1 interferon in response to viral infections [5, 6]. On the other hand, bone marrow-derived DCs (myeloid DCs) are present in most tissues and are characterized by coexpression of CD11c and CD11b markers. As reviewed by Breckpot et al. (2009), these DCs respond to GM-CSF and are capable of producing IL-12 in response to toll-like receptor ligands. Interestingly, DCs have been shown to possess a remarkable cellular plasticity. For example, pDCs could acquire myeloid DC characteristics under the influence of viral infection .
In order to elicit productive T cells responses, DC major histocompatibility (MHC)/peptide complexes must interact with specific T cell receptors (Signal 1) in the context of an appropriate costimulatory molecule interaction between both cell types (Signal 2). It has been recently considered that the microenvironment where this interaction occurs (Signal 3) will determine the fate the subsequent immune response towards an immunogenic or tolerogenic response . A clear example of the relevance of the microenvironment on DC biology can be observed in tumor settings. Molecules present in the tumor milieu such as vascular endothelial growth factor (VEGF), interleukin (IL)-10 and prostaglandin-2 (PGE-2) can profoundly affect the biology of DCs making them immunosuppressive, incapable of inducing specific immune responses or capable of inducing regulatory T cells [7, 8]. In particular, DCs showing low levels of costimulatory molecules have been detected in microenvironments characterized by high levels of VEGF . These DCs, showing highly immunosuppressive properties, are able to render T cells anergic or tolerised, thus abrogating immune responses. On the contrary, endothelial cell-produced antiangiogenic cytokine vascular endothelial growth inhibitor induces DC maturation . Furthermore, treatment of the tolerogenic DCs with inflammatory molecules, render immunogenic DCs with the capability to activate T cells . Besides an immune "paralysis", we and others have shown that DCs, or leukocytes expressing DC markers are able to produce angiogenic factors and can promote angiogenesis [12–15].
We hypothesized that this plasticity might be caused not only by the action of specific cytokines or growth factors, but also by the interaction of these cells with extracellular matrix (ECM) components. Herewith, we performed a series of studies in order to determine the influence of different surfaces and growth factors on the biological properties of myeloid DCs.
Six to eight week old female C57BL/6 (H-2Kb) and BALB/c (H-2Kd) mice (Charles River Laboratories, Wilmington, MA) were used in protocols approved by the Institutional Animal Care and Use Committee at Ohio University.
In vitro generation and maturation of murine DCs
Murine DCs were generated from bone marrow precursors recovered from femurs and tibiae of 6-8 week old female C57BL/6 mice by the method of Lutz et al.[16, 17]. Briefly, bone marrow cells were dispersed by vigorous pipetting and cultured in RPMI-1640 supplemented with penicillin (100 μg/ml), streptomycin (100 U/ml), L-glutamine (2 mM) and 10% heat-inactivated fetal bovine serum (FBS) (all Invitrogen, Carlsbad, CA) in the presence of 20 ng/ml of recombinant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF, 315-03, Peprotech Inc., Rocky Hill, NJ) for 8 days. GM-CSF was replenished on days 3 and 6. In some experiments, maturation was induced by culturing the cells for 2 days in the presence of 5 ng/ml GM-CSF, 20 ng/ml mouse tumor necrosis factor alpha (TNF-α, 315-01A, Peprotech) and 100 ng/ml bacterial lipopolysaccharide (LPS from E. coli, serotype 0111:B4, L2630, Sigma).
Cell lines and tumors
In some experiments we used the murine ID8-Vegf-A cell line of ovarian cancer . The ID8 cell line is a tumor cell line derived from spontaneous in vitro malignant transformation of C57BL/6 mouse ovarian surface epithelial cells originally generated by Roby et al.. This line has been engineered to express high levels of VEGF-A (VEGF-164) . These cells were maintained in DMEM supplemented with 2 mM L-glutamine, 100 μg/ml penicillin, 100 U/ml streptomycin, and 10% heat-inactivated fetal bovine serum (FBS) (all Invitrogen). Ectopic ID8-Vegf-A solid ovarian tumors were initiated in C57BL/6 mice by subcutaneous injection of 7 × 106 tumor cells [20, 21].
Culture of DCs on different surfaces
In order to investigate the effect of different surfaces on the biology of DCs, these cells were seeded on commercially available 6-well plates coated with different extracellular matrix components such as fibronectin, collagen I, gelatin, Matrigel or synthetic poly-D-Lysine (all BD Biosciences, San Jose, CA). Controls included cells cultured on regular polystyrene tissue culture plates (Corning Costar, Corning, NY). DCs were seeded on these plates at a concentration of 5 × 105 cells/ml in either RPMI 10% FBS or endothelial EBM-2 medium with supplements (EGM2-MV BulletKit, Lonza) with or without the addition of GM-CSF (3 ng/ml). Cells were cultured up to 3 weeks in these conditions and media was replenished once a week. Pictures of live cells were obtained with an inverted microscope attached to a Motic 2000 Camera (Motic, Richmond, British Columbia, Canada). In order to perform specific studies, after different times in culture cell supernatants were collected, surfaces washed with PBS and attached cells recovered by using cell scrapers.
In order to investigate the effect of tumor-derived GM-CSF on DCs, we prepared tumor conditioned medium. To accomplish this, ID8-Vegf-A cells were cultured until 80% confluence and supernatants collected and filtered. Then, these supernatants were mixed with RPMI medium (30:70; conditioned medium:RPMI) and used to culture DCs for 1 week. As a control of GM-CSF specificity we also cultured these cells in the presence of anti-GM-CSF receptor antibody (rabbit polyclonal, sc25472, Santa Cruz Biotechnology Inc. Santa Cruz, CA) as previously described  or isotype control.
Purification of CD11c by means of magnetic sorting
In some experiments CD11c cells were recovered from the cultures by magnetic sorting. To accomplish this, cells were mechanically detached from the culture plates by using cell scrapers. After blocking Fc receptors with anti-CD16/CD32 antibody (Fc block, 2.4G2; BD Pharmingen, San Diego, CA), cells were labeled with anti-CD11c magnetic beads (MACS Miltenyi, Auburn, CA) and positive cells isolated by using MS paramagnetic columns in an octoMACS magnet (all MACS Miltenyi) following the manufacturer's instructions.
Cells were subjected to three-color flow cytometry on a FACSort flow cytometer using CellQuest 3.2.1f1 software. (Becton Dickinson, San Jose, CA). We collected 10.000 events per sample. Non-specific staining was blocked with Fc block in FACS buffer (PBS with 2% FBS and 0.05% sodium azide). Fluorochrome-conjugated monoclonal antibodies against CD45 (30-F11), CD11c (HL3), CD80 (16-10A1), CD86 (GL1), MHC-II (KH74), CD54 (3E2), CD11b (M1/70), GR1 (RB6-8C5), CD40 (3/23), PSGL-1 (2PHI), (all BD Biosciences, San Diego, CA); CD29 (HMb1-1), CD49b (DX5), CD49d (R1-2), CD49e (HMa5-1), CD49f (GoH3), CD41 (MWReg30), CD51 (RMV-7), CD61 (2C9.G3), OX40-L (RM134L), CD44 (IM7), CD18 (M18/2), (all eBioscience, San Diego, CA); and CD49a (HMalpha1, AbD Serotec, Raleigh, NC) were used at 1/100 dilution.
RT-PCR and Real-Time Quantitative Reverse Transcription-PCR
RNA was isolated with TRIzol (Invitrogen) and then reverse transcribed by using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) following the manufacturer's instructions. All RNA samples were treated with DNAse in order to eliminate possible contaminating genomic DNA. For qualitative PCR analysis, the PCR cycling was conducted with Taq polymerase at 94°C (30 s), 57°C (30 s), and 72°C (20 s) for 40 cycles. Expression of specific molecules was also analyzed at the level of RNA by means of quantitative real time RT-PCR (qPCR) analysis. For qPCR experiments, we used the absolute quantification method by generating standard curves for our genes of interest, and housekeeping gene. We normalized the cDNA load to mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data were expressed as relative units to GAPDH mRNA molecules. In these assays, we used (PerfeCTa SYBR Green FastMix, Quantas Biosciences) for detection of the PCR reaction. Each amplification experiment was performed in 96-well optical grade PCR plates covered with optical film) in an iCycler iQ5 real-time PCR instrument (Bio-Rad Laboratories, Hercules, CA).
List of primers used for qualitative and quantitative PCR studies.
Forward 5'-CAA ATG CCA AAT CTT GCG GAG A-3'
Reverse 5'-GCA TTC CTT CTT GCA AAA ATG TCG T-3'
Forward 5'-GAG AGA GAG ATC AAT GCC TTG TGT GAA-3',
Reverse 5'-CGG ATT GGT GAC TAA AGT TGA TCC AAA-3'
Forward 5'-CCC TGT GGA CCT ACC CAC TGC-3',
Reverse 5'-TGA GGG TCA ATC CCA GGC TCA-3'
Forward 5'-GGG AGC CTG TCG GAC CAC AT-3',
Reverse 5'-AAA GCG CAG GGT CCA ACA CA-3'
Forward 5'-TGG AGA AAA TTT TGC ATC ATG TCA AGC-3',
Reverse 5'-CGG TGC CAG TCC AGT ACG ATG-3'
Forward 5'-TTG GGG GAC CTG GAC CAA GA-3',
Reverse 5'-TGG GCC TCC CGG GAA TAT AAA-3'
Forward 5'-CGG GAG CCT CTT CGG CTT CT-3'
Reverse 5'-CTG CAG CGG GAG TGC TTC TG-3'
Forward 5'-GTC GCG CCA ACA CCA TGA G-3'
Reverse 5'-GGG TCA CCG CCA AGC TGA AG-3'
Forward 5'-CCA CCA GTG GTT TGG AGC CTC T-3'
Reverse 5'-TCC AAC TGG CTC TCT CTC CTG CT-3'
Forward 5'-TGA CGC CAT CAT GCA GGC TA-3'
Reverse 5'-TGG GTC TTG GCA TCC GTG GT-3'
Forward 5'-TGT GTG CCA ACC GGT ACC TT-3'
Reverse 5'-TTC CAG TCG TTC AAA GAA GAA ACA-3'
Forward 5'-CAT GCC TGT CAC GTT GAA TGA AGA-3'
Reverse 5'-TCA GGC GGG TCT GCA CAC AT-3'
Forward 5'-CCT GCA CCA CCA ACT GCT TA-3'
Reverse 5'-CAT GAG TCC TTC CAC GAT ACC A-3'
Forward 5'-GGG GCC GGA TGG ATT ACT TT-3'
Reverse 5' -CCA TGA AAA ACC CGT CTC CA-3'
Forward 5'-GGG ACG GTA TCC ATC ACT AAG A-3'
Reverse 5'-CTT TAC CGC GAT AGC TCG AA-3'
Forward 5'-GCA TCG CTC AGA TCC GTG GT-3'
Reverse 5'-GAA TGT GGC CAC CAG CAA GG-3'
Forward 5'-TAA AGG CCG CTC GGA TGG TT-3'
Reverse 5'-CCA ACT ACG GTC GCG TCC AC-3'
Forward 5'-CCT AGC TGG GTT GGC TGT GCA T-3'
Reverse 5'-GCT GCG ACC CCA CAC TTC GT-3'
Forward 5'-CCG AGC TAT TGC AGC CCA TT-3'
Reverse 5'-GCC ACT CAC TGT CCC ATC CA-3'
Forward 5'-GCC AGC ACA TAG AGA GAA TGA GC-3'
Reverse 5'-CAA GGC TCA CAG TGA TTT TCT GG-3'
Forward 5'-CGC CTG CTG CTT GTT GCA CT-3'
Reverse 5'-TCC ATG GCA CCA CTT TCT TCT GG-3'
Forward 5'-TCA TGC AAG CAG GCC AGA CTC TC-3'
Reverse 5'-CCT TTT GTC CTC CTG GCT CAC G-3'
Forward 5'-GGG CTT GAT TTC ACC TGGC ACT C-3'
Reverse 5'-CGC CAC AGT CCC AGG AAA GG-3'
Solid tumor samples were snap-frozen in OCT medium (Tissue Tek, Sakura, Torrance, CA) and sections were prepared using a Leica CM1950 Cryostat (Leica Microsystems, Bannockburn, IL). Sections were fixed in cold acetone for 10 minutes, pretreated with 3% H2O2 for 20 min to block endogenous peroxidase activity and blocked in normal horse serum (Vector Laboratories). Biotinylated rat anti-mouse CD11c (HL3) and biotinylated hamster isotype control (both BD Pharmingen) were used at 1:50 dilution for these studies. Then, the Vectastain ABC kit was applied as described by the manufacturer (Vector Laboratories). Sections were counterstained with Gill's hematoxylin (Vector Laboratories). Images were acquired through a Micropublisher 5.0 Digital CCD Color Camera (Qimaging, Surrey, BC Canada).
The concentration of different cytokines in culture supernatants was quantified by antigen capture ELISA. We used the following purified antibodies for capture: anti-mouse IL-1α (ALF-161), IL-6 (MP5-20F3) (both eBioscience, San Diego, CA) and anti-mouse-VEGF (BAF493, R&D Systems). For detection, we used biotin anti-mouse IL-1 α (Polyclonal), IL-6 (MP5-32C11) and biotin anti-mouse VEGF (AF-493-NA, R&D Systems) at 1 μg/well. Standard curves were constructed using recombinant murine IL-1α (220-11), IL-6 (216-16) and VEGF (450-32) (all Peprotech). Murine FGF was evaluated in culture supernatants by using the Human FGF-basic ELISA Development Kit (Peprotech) which cross-react 100% with mouse. Each dilution of recombinant standard or sample was assayed in duplicate. The reaction was developed by using streptavidin-horseradish peroxidase (554066, BD Pharmingen) and the 2,2'-azino-di-[3-ethylbenzthiazoline sulfonate(6)] (ABTS) substrate system (Roche Diagnostics GmbH, Mannheim, Germany). The blue-green color produced by enzymatic activity was quantified at 405 nm in an ELISA microplate reader (Multiskan RC, ThermoLabsystems).
Murine myeloid C57BL/6 DCs recovered from long-term cultures and treated for 48 h with an inflammatory cocktail as described above were seeded in 96-well round-bottom plates at a concentration of 1 × 105/well in RPMI containing 10% FBS. Spleens were resected from healthy BALB/c mice and minced in a sterile fashion to yield a single cell suspension and erythrocytes were eliminated by hypotonic shock. Then untouched CD3 T cells were purified from this suspension by magnetic sorting using the Pan T Cell Isolation Kit (MACS Miltenyi) following the manufacturer's instructions. T cells were labeled with CFSE as previously described  and were incubated at a concentration of 1 × 105 cells/well with the recovered DCs for 5 days. CFSE dilution, an indication of cell proliferation, was assessed by qualitative flow cytometry analysis of gated CD3 T cells.
For multiple comparisons we performed ANOVA analysis with post-analysis by the Tukey-Kramer multiple comparisons test or the Dunnett's post comparison test. A value of p < 0.05 was considered significant. Data are expressed as mean ± SD. Data was analyzed by using the Graph Pad Instat software (GraphPad Software, Inc., San Diego, CA).
Expression of adhesion molecules by myeloid DCs
Murine myeloid(m) DCs have been extensively used in order to determine the efficacy and improvement of DC-based vaccines; investigate DC:T cell interactions or DC development; and determine their role in pathological conditions such cancer or infectious diseases [29–36]. Thus, we decided to use them as a model for our studies. We have previously reported that these cells exhibit high plasticity, being capable of acquiring angiogenic properties in vivo under pathological conditions . We hypothesized that this might be caused not only by the presence of specific cytokines or growth factors in their microenvironment, but also by their physical interaction with different surfaces such as those generated by deposition of extracellular matrix (ECM) components. To investigate this we performed a series of studies in order to determine the influence of adhesion surfaces on the biology of DCs. First, by means of qualitative PCR analysis we studied at the level of RNA the expression of adhesion molecules capable of interacting with ECM components. In particular we investigated the expression of CD29 (binds with CD49 isoforms to generate adhesion complexes), CD49a (CD49a\CD29 complex binds to laminin and collagen), CD49b (CD49b\CD29 complex binds to collagen and laminin), CD49c (CD29\CD49c complex binds to laminin, collagen, fibronectin and thrombospondin), CD49d (CD49d\CD29 complex binds to fibronectin, and cellular components VCAM and MadCam), CD49e (CD49e\CD29 complex binds to fibronectin), CD49f (CD49f\CD29 complex binds to laminin), CD41 and CD61 (CD41/CD61 complex binds to fibrinogen, fibronectin and vitronectin), and CD51 (CD51/CD61 complex mediates adhesion to fibrinogen, fibronectin, vitronectin and thrombospondin) . As shown in Figure 1C, we were able to detect expression of these molecules in mDCs at the level or RNA. In a series of complementary studies, we decided to investigate the expression of these molecules at the level of protein. To accomplish this we performed a direct staining of surface mDC molecules using fluorescent antibodies and we analyzed the cells qualitatively by means of flow cytometry. As shown in Figure 1D, immature mDCs express detectable levels of surface CD29, CD49d, CD49e, CD49f and CD51 at the level of protein when compared to the isotype controls. Contrary to what we observed at the level of RNA, we detected very low to null expression of CD49a, CD41 and CD61 in the same cells by flow cytometry analysis using direct staining. We cannot discard that these molecules are expressed at very low levels on the surface of the cells and require more sensitive methods, such as indirect staining, for their detection by flow cytometry.
Generation of DC cultures on different surfaces and media
We have previously shown that murine mDCs could be cultured on Matrigel in the presence of tumor factors for long periods of time . Similar studies were performed with human DCs grown on fibronectin and treated with tumor factors . In order to investigate the role of different surfaces on the biology of mDCs, we cultured these cells on plates coated with fibronectin, collagen I, gelatin, Matrigel, poly-D-Lysine (a synthetic polymer which displays uniform net positive charges on the adhesion surface), or regular plastic culture surfaces (polystyrene) for up to 3 weeks. Also, to determine the effect of different environmental conditions, we cultured these cells in regular DC medium (RPMI) or endothelial cell growth medium (EGM). Regular DC medium consisted of RPMI medium supplemented with 10% FBS. The EGM endothelial medium is supplemented with 10% FBS together with several growth factors such as (human (h)EGF, hydrocortisone, hVEGF, hFGF-B, hIGF-1, ascorbic acid) that support endothelial cell proliferation.
Phenotypical analysis of DCs upon culture on different surfaces and media
Production of angiogenic factors by 1-week mDC cultures
Finally, DCs cultured for one week on different surfaces and media conditions express CD54 as determined by comparison with isotype controls in a qualitative flow cytometry assay (Figure 7C). This molecule has a crucial role in the clustering of DCs with lymphocytes by interacting with LFA-1 on the surface of T cells . This indicates that these mDCs are able to interact with T cells although not being capable of positively activating them due to low levels of costimulatory molecules.
Relevance of GM-CSF on the phenotype of DC cultures
Generation of GM-CSF by ovarian cancer cells and its effect on mDC phenotype
Similarly, blocking the effect of tumor-derived GM-CSF decreased the levels of costimulatory molecules in these cells, as determined when comparing the proportion of cells expressing both markers in anti-GM-CSFR treated and control samples. This indicates that the presence of this molecule in the tumor microenvironment might help preserve mDC function.
Angiogenic properties of long-term DC cultures
Immunological capabilities of DCs upon long-term cultures
To further determine the immune capabilities of these cells, after stimulation with an inflammatory cocktail we cocultured them with CFSE-stained allogeneic BALB/c T lymphocytes. Consistently with the ELISA and FACS data, CD11c cells recovered from Matrigel and poly-D-Lysine cultures induce higher levels of proliferation of allogeneic T cells than cells recovered from other conditions (Figure 16D). Indeed, CD11c recovered from collagen cultures, which produce the lowest levels of inflammatory cytokines upon stimulation, were unable to induce proliferation of T cells as determined by qualitative flow cytometry analysis. Altogether, these data indicate that the interaction with different surfaces affect in different ways the immunological properties of mDCs.
DCs present in peripheral tissues sample the organism for the presence of antigens, which they take up, process and present in their surface in the context of MHC molecules. Typically, antigen-loaded DCs migrate to immunological organs where they present the processed antigens to T lymphocytes thus triggering specific immune responses. On the other hand, a role of DCs as promoters of angiogenesis under pathological conditions has been suggested [13, 15, 58, 59]. Our hypothesis is that in the tumor microenvironment, this phenotypic shift is caused by the combined effect of cytokine/growth factor signaling and interaction with ECM components.
We and others have shown that monocytes or DCs can undergo an endothelization process in vitro characterized by the loss of CD14/CD45 and upregulation of endothelial markers such as CD31, CD34, von Willebrand factor, VEGF receptor (VEGFR)-2 and VE-Cadherin [12, 41, 60]. These cells can display other characteristics of bonafide endothelium such as LDL uptake, lectin binding or formation of cord-like structures in 3D gels [12, 24, 41, 58, 60, 61]. DCs can also promote angiogenesis by generating angiogenic factors, i.e., DCs with proangiogenic properties have been described in tumors and other pathological conditions [15, 58, 62–64]. These data indicate that DCs have a high plasticity, reacting to their microenvironment by changing their morphology and biological activity. This becomes important taking into account that it has been postulated that the microenvironment where they encounter their antigenic stimuli will define the outcome of the DC:T cell interaction (Signal 3) and the consequent immune response.
Several reports indicate that soluble factors can alter the biology of DCs. Tumor-associated cytokines such as VEGF, IL-10 and PGE-2 can profoundly affect the nature of APCs. In particular, DCs showing low levels of costimulatory molecules have been detected in tumors expressing high levels of VEGF  and tumor patients treated with anti-VEGF antibody showed a decrease in the levels of immunosuppressive DCs . In the same way, it has been shown that the endothelial cell-produced antiangiogenic cytokine vascular endothelial growth inhibitor induces DC maturation . Another molecule present in the tumor microenvironment that can profoundly affect DC biology is PGE-2. This molecule has been shown to induce the production of IL-10 and VEGF by human DCs .
On the other hand, few studies have been undertaken in order to determine the effect of different ECM components on the biology of DCs. Foundational work on this subject was performed on 1998 by Brand et al.. This research group, working with human monocyte-derived DCs, investigated the adhesion of these cells to collagen I, IV and fibronectin. They were able to show that these cells adhere differentially to these ECM components, rapidly changing their morphology when they attached to fibronectin or increasing their maturation status upon interaction with collagen I. In another series of experiments, Ammon et al. described the expression of different integrin receptors in human monocytes, monocyte-derived DCs and macrophages. More recently Kohl et al.,  reported that human DCs derived from CD34 precursors or from monocytes differentially bind to ECM components, and display different expression of surface integrins.
Herewith we report that attachment to a surface together with factors provided in the media allowed long-term mDC cultures to exhibit different immunological capabilities. DCs are usually considered short-lived, terminal cells whose main function is to activate T cells in order to induce specific immune responses. We were able to demonstrate that these cells need the presence of GM-CSF in order to be able to establish long-term cultures; that GM-CSF can be produced by ovarian cancer cells and that is expressed in the microenvironment of mouse ovarian tumors. In terms of the role of adhesion surfaces on the biology of these cells, we were able to show that cells cultured on different surfaces showed expression of angiogenic molecules such as HGF, bFGF, MMP2 and MMP9. We also showed that these cells are able to produce VEGF-A, -B and PIGF. Taking into account that most of these molecules were expressed in every condition it is tempting to speculate that if mDCs are not driven to initiate an immune response they will by default remain in a proangiogenic state. Further, since we were able to detect the expression of VEGFR-1 and -2 in these cells it is tempting to speculate that some of these angiogenic molecules might act in an autocrine fashion, promoting cell survival or induction of other angiogenic molecules. Finally, expression of MMPs by DCs might promote VEGF availability  thus contributing to angiogenic process but might also help degrade ECM components facilitating metastasis in a tumor setting.
In terms of immunological properties, we observed that after 1 week culture in non-inflammatory conditions, mDCs showed low levels of costimulatory molecules. Attachment to the different surfaces did not inhibit the capability of these cells to respond to inflammatory stimuli. In particular, these cells upregulated the expression of costimulatory molecules, and produced IL-1α, IL-6 and nitric oxide when cultured in the presence of LPS and TNFα. This effect was less pronounced when cells were cultured with EGM, indicating that the factors present in this medium can decrease the effects of inflammatory factors. Further, DCs cultured on different surfaces for 3 weeks showed differential immunological capabilities. In particular, DCs recovered from Matrigel, (a gelatinous protein mixture purified from murine tumor stroma) and from poly-D-Lysine (an artificial polymer) were able to respond to maturation stimuli similarly to fresh mDCs. Upon stimulation, these cells produced large quantities of IL-1α and IL-6, and induced proliferation of allogeneic lymphocytes. On the other end, DCs recovered from collagen I surfaces were completely unresponsive to maturation stimuli.
Herewith we demonstrated that the combination of soluble factors together with adhesion surfaces determines particular mDC profiles. Thus, in order to design DC-based vaccines or treatments focused on changing the phenotype of DCs associated with diseases, such as cancer [68–70] or atherosclerosis [71, 72] among others, it becomes necessary to fully investigate the microenvironment in which these cells are present or will be delivered.
This work supported in part by the NIH under Grant R15 CA137499-01 (F.B.) and a startup fund from Ohio University (F.B.).
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