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Tumor Necrosis Factor Alpha-Stimulated Endothelium: An Inducer of Dendritic Cell Development from Hematopoietic Progenitors and Myeloid Leukemic Cells

^a Institute for Transfusion Medicine, Charité, Universitätsmedizen Berlin, Berlin, Germany;
^b Weill Medical College of Cornell University, New York, New York, USA;
^c Memorial Sloan-Kettering Cancer Center, New York, New York, USA

Key Words. CD34⁺ cell • Dendritic cell • Endothelial cell • Cytokines • Apoptosis

Anja Moldenhauer, M.D., Charité, Universitätsmedizin Berlin, Institute for Transfusion Medicine, Augustenburger Platz 1, 13353 Berlin, Germany. Telephone: 49-160-1090837; Fax: 49-30-450-553988; e-mail: anja.moldenhauer{at}charite.de

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Discussion Conclusion References

 
Especially when exposed to inflammatory stimuli, endothelial cells (EC) have been shown to promote the maturation of monocytes into dendritic cells (DC) and the long-term proliferation of CD34⁺ cells by constitutive cytokine production and direct cellular contact. We therefore hypothesized that cytokine-stimulated EC would induce hematopoietic progenitor cells to develop into mature dendritic cells. To test this theory, human CD34⁺ cells derived from cord blood or leukapheresis products were cultured with a monolayer of either interleukin (IL)-1ß, IL-4, or tumor necrosis factor (TNF)--stimulated human umbilical cord EC. The cells in suspension were analyzed weekly over a period of 6 weeks. IL-1ß supported cell expansion, whereas IL-4 had no effect on cell expansion or DC differentiation. Only TNF--stimulated EC induced the development of mature, allostimulatory DC with a high expression of CD83, HLA-DR, CD1a, and costimulatory molecules like CD80 and CD86. Acute myeloid leukemia cells from the cell line Kasumi-1 also developed DC-like features when cocultured with TNF--stimulated EC. Direct contact between endothelial and progenitor cells increased the number of developing DC. Cell cycle analysis and apoptosis studies demonstrated a reduced G₂M fraction, an increased S fraction, and a decrease in TNF--dependent apoptosis of DC developing in the presence of endothelial cells. As shown by electron and confocal microscopic studies, intimate interactions between EC and DC occurred, resulting in the internalization of the developing DC within the EC monolayer and a bidirectional exchange of proteins. We conclude that, via the action of TNF-, inflamed human endothelium can induce CD34⁺ and leukemic cells to differentiate into dendritic cells.

	INTRODUCTION

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Dendritic cells (DC), the most potent antigen-presenting cells, serve as a nexus between immunogenicity and tolerogenicity [1, 2]. Two pathways of DC differentiation have been extensively studied in regard to their clinical applicability: A) differentiation of peripheral blood monocytes into DC after exposure to various maturation stimuli [3, 4], and B) differentiation of CD34⁺ hematopoietic progenitors [5–7] and leukemic cells into DC [8]. While monocytes can differentiate into DC within 48 hours [9], CD34⁺ cells usually need 7–12 days [5, 10].

Endothelial cells (EC) have been demonstrated to recruit granulocytes [11], lymphocytes [12], and monocytes [13], the latter even differentiate into DC while transmigrating through endothelium [14, 15]. Furthermore, EC also support the proliferation of CD34⁺ cells by constitutive production of cytokines and direct cellular contact [16, 17]. Little is known about the function of CD34⁺ cells at the site of inflammation. Less than 0.1% of peripheral blood mononuclear cells are CD34⁺ hematopoietic progenitors [18]. Besides, CD34⁺-derived DC have been shown to be less allostimulatory than monocyte-derived DC [19].

Given these facts, the role of circulating CD34⁺ hematopoietic progenitors in primary immune defense seems to be of minor importance, although CD34⁺ cells are mobilized in an inflammatory response due to the release of various cytokines, e.g., G-CSF, macrophage-CSF (M-CSF), interleukin (IL)-1, and stromal cell-derived factor-1 [20–24]. However, in contrast to quiescent monocytes, hematopoietic progenitors have the potential to proliferate, thereby offering an additional source of replicating dendritic cells. Moreover, their superiority in activating antigen-specific CD8⁺ T cells has been demonstrated previously [25].

We therefore hypothesized that the presence of inflamed endothelium simulated by stimulating a monolayer of EC with tumor necrosis factor (TNF)-, IL-1ß, or IL-4 would induce CD34⁺ cells to develop into dendritic cells. To test this hypothesis, we established a CD34⁺/endothelial cell coculture system that allowed us to demonstrate the influence of these cytokines and to elucidate the role of direct cell-to-cell contact in DC production. Furthermore, we observed that myeloid leukemic cells, represented by the acute myeloid leukemia (AML) cell line Kasumi-1, differentiated into DC in the presence of TNF--treated EC.

	MATERIALS AND METHODS

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Media and Antibodies
The basic medium consisted of Iscove’s modified Dulbecco’s medium supplemented with 20% fetal bovine serum (FBS), 2 mM L-glutamine, 50 µg/ml gentamicin, and 7.3 x 10^-5 monothioglycerol. The endothelial cell-conditioning medium consisted of M199 with 16% FBS, 4% human serum, 2 mM L-glutamine, 0.15 mg/ml endothelial growth factor supplement (Intracel; Rockville, MD; http://www.intracel.com), 0.015 mg/ml heparin, and 1% fungicide. The AML cell line Kasumi-1 (kindly provided by N. Kamada, Hiroshima University, Japan) was maintained in RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, and 100 U/mL penicillin/ streptomycin.

All monoclonal antibodies used for flow cytometry were directly conjugated with either phycoerythrin (PE) or fluorescein isothiocyanate (FITC). Dendritic cells were detected using CD83-PE (Immunotech; Marseilles, France; http://www.immunotech.fr), CD1a-PE (Becton Dickinson; San Jose, CA; http://www.bd.com), CD80-PE (Pharmingen; San Diego, CA; http://www.bdbiosciences.com/pharmingen), CD86-FITC (Pharmingen), and HLA-DR-FITC (Becton Dickinson). Monocytic cells were detected using CD14-FITC (Immunotech) and T cells were detected by CD3-FITC (Pharmingen). Developing DC were discerned from EC using CD45-FITC (Pharmingen), CD45-PE (Becton Dickinson), and CD33-PE (Becton Dickinson). For confocal microscopy, both nonconjugated anti-human CD45 and murine anti-vascular adhesion molecule (VCAM)-1 IgG were used as primary antibodies (Pharmingen).

Purification of Human CD34⁺ Cells
Human CD34⁺ cells were isolated from discarded samples of frozen leukapheresis products collected from patient peripheral blood after stem cell mobilization or from cord blood specimens as described by Frey et al. [26]. All human samples were collected according to guidelines approved by the Institutional Review Boards of Memorial Sloan-Kettering and Cornell University Medical College.

Separation of EC and Bone Marrow Fibroblasts
Human umbilical cord EC were harvested by flushing the umbilical vein with collagenase as described by Jaffe et al. [27] and grown for 5–10 days in flasks containing EC conditioning medium. Bone marrow cells from healthy volunteers were obtained as described by Castro-Malaspina et al. [28]. Briefly, marrow stroma cells aspirated from the posterior superior iliac crest were separated by velocity sedimentation and adherence and cultured in basic medium in T-25 or T-75 tissue culture flasks. After two to three passages, the cultured EC and fibroblasts were stored in liquid nitrogen with 10% dimethyl sulfoxide in EC conditioning medium or basic medium. For the experiments, EC and marrow stroma cells were recultured to confluence and the fibroblasts were irradiated with 30 Gy prior to the addition of CD34⁺ cells.

Coculture System
CD34⁺ hematopoietic progenitor cells (10⁵) derived from cord blood (n = 6), leukapheresis products (n = 7), or Kasumi-1 cells (n = 7) were cocultured on 6-well plates with a confluent monolayer of human umbilical cord EC. The CD34⁺ cells were plated either in direct or indirect contact with the EC. In the latter case, they were placed on top of a 0.4 µm microporous transmembrane above the EC, thus forming a transwell culture system (noncontact group). Every other day, 0.5 ml of supernatant was removed and replaced with fresh basic medium or EC conditioning medium plus cytokines. CD34⁺ and Kasumi cells cultured on nonstimulated EC served as the controls. The DC induction capacities of the following cytokines were investigated: TNF- (20, 50, or 100 ng/ml, specific activity 1 x 10⁸ U/mg; GenenTech; San Francisco, CA; http://www.gene.com), IL-1ß (100 U/ml, specific activity 3 x 10⁸ U/mg; Syntex Laboratories; Palo Alto, CA), and IL-4 (20 ng/ml, specific activity 1 x 10⁷ U/mg, Intergen; New York, NY; http://www.intergen.com). In two experiments, CD34⁺ progenitors were cultured on marrow stroma cells with and without TNF-.

To compare the allostimulatory capacity of DC generated in contact to EC with that of DC generated by cytokine-stimulated CD34⁺ cells [5, 6], CD34⁺ cells were cultured in a cytokine cocktail containing TNF- (100 ng/ml), GM-CSF (100 ng/ml, specific activity 2.5 x 10⁷ U/mg; Immunex; Seattle, WA; http://www.immunex.com), c-kit ligand (20 ng/ml stem cell factor, specific activity 5 x 10⁶ U/mg; Amgen; Thousand Oaks, CA; http://www.amgen.com), and IL-4 (20 ng/ml).

Cell Count and Flow Cytometry
Every 7 days, the cells in suspension were counted by hemocytometry, and the number of DC was determined by flow cytometry using monoclonal antibodies directed against DC-typical antigens such as CD83, CD1a, CD80/86, and HLA-DR. Flow cytometry and apoptosis studies were performed as described previously [29]. Developing DC adhering to the EC monolayer were removed by incubating the samples with 200 µl 0.1% cold EDTA in Hank’s buffered salt solution until the attached cells started to detach (1–2 minutes); this process was carefully controlled under a phase-contrast microscope. The loosened cells (contact-attached) were analyzed parallel to the cells in supernatant (contact-detached) and EC. The latter were harvested in 1 ml phosphate-buffered saline (PBS) after additional incubation with 200 µl 0.1% EDTA. Kasumi cells in a transmembrane (noncontact), TNF--stimulated, and unstimulated Kasumi cells (no EC) were assessed and compared. Apoptosis and cell cycle analyses were performed after 24 and 48 hours of culture.

T-Cell Proliferation
Proliferation of allogeneic T cells was analyzed by ³H-thymidine incorporation in mixed lymphocyte reactions as described previously [29]. Briefly, aliquots of cultured DCs were harvested on day 15, irradiated with 30 Gy gamma radiation and incubated in graded concentrations (stimulator to responder ratio [S/R] 1:30 to 1:480) with 1.5 x 10⁵ allogeneic human T-cells in 96-well flat-bottom tissue culture plates (0.2 ml medium/well). Proliferation was measured by ³H-thymidine (1 µCi/well, Amersham Pharmacia Biotech; Piscataway, NJ) added for 16 hours on day 5 of culture.

Adenoviral Vector
The adenoviral vector, an E1^-E3^- replication-deficient, serotype-5 adenoviral vector that contains the green fluorescent protein (GFP) reporter gene driven by a cytomegalovirus early-intermediate promoter enhancer in the E1 position [26], was kindly provided by Neil Hackett, Gene Core Facility Unit of the New York Hospital. Viral particle concentrations were determined by the absorbance at 260 nm and the extinction coefficient for adenovirus of 9.09 x 10^-12 ml per particles and cm [30]. Stocks were titrated on 293 cells (American Type Culture Collection; Rockville, MD; http://www.atcc.org) by plaque assays, and titers were expressed as infectious particles of plaque-forming units (pfu) per ml. The multiplicity of infection (MOI) was calculated as pfu per target cell.

Morphology and Electron and Confocal Microscopy
Phase-contrast microscopy of liquid cultures and light microscopy of cytospin preparations were carried out as previously described to document the morphology and maturation features of the investigated cells [29]. For cocultures studied by electron microscopy, EC were grown in 24-well plates on inserted coverslips (Thermanox; Nunc; Wiesbaden, Germany; http://www.nuncbrand.com) to confluence prior to the addition of progenitor cells with or without TNF-. Twenty-four to 48 hours later, the slips were removed and fixed in a mixture of 4% paraformaldehyde/2.5% glutaraldehyde/0.02% picric acid in 0.1 M sodium cacodylate buffer (pH 7.3). After rinsing the cells with 0.06 M phosphate buffer, samples for scanning electron microscopy were prepared [31, 32] and investigated using a DSM 982 Gemini (Zeiss; Oberkochen, Germany; http://www.zeiss.com). For transmission electron microscopy the samples were postfixed with 2% OsO₄ for 2 hours, rinsed three times with phosphate buffer, dehydrated in a graded alcohol series and embedded in araldite. Thin sections for transmission electron microscopy were prepared and examined using an EM 906 (Zeiss; Jena, Germany).

In samples studied by confocal microscopy, a confluent monolayer of EC was transduced for 2 hours with an adenoviral vector encoding for GFP (MOI 100). Remaining vectors were removed by washing the samples twice with FBS. Two days later, when more than 80% of the EC had turned green, the cells were replated on chamber slides (Lab-Tek^TM; Nalge Nunc; Rochester, NY; http://www.nalgenunc.com) coated with 0.2% gelatin (J.T. Baker; Phillipsburg, NJ; http://www.jtbaker.com) that was dissolved in distilled, demineralized water. Within 24 hours, the cells had formed a confluent monolayer in the center of each chamber. Kasumi-1 cells were seeded on top of TNF--stimulated and unstimulated, green-fluorescing EC. The supernatants were removed 24–48 hours later, and the samples were washed twice with PBS and fixed in 100% methanol at -20°C for 20 minutes. Next, the samples were incubated with murine anti-human CD45 antibodies (10 µg/ml, Pharmingen), washed twice with PBS, and stained with a secondary rhodamine-conjugated goat anti-mouse IgG (10 µg/ml). Permount (Fisher Scientific; Pittsburgh, PA; http://www.fisherscientific.com) was used as the mounting medium. Pictures were taken using a laser scanning microscope 510 and evaluated by the confocal microscopy software v2.5 (Zeiss). For comparative analysis, Kasumi-1 cells were incubated with TNF- for 2 hours before adenoviral transduction with GFP (MOI 500 [33]) and seeded on top of a monolayer of EC 24 hours later (i.e., after 50% of the Kasumi cells fluoresced green). In this experiment, anti-VCAM-1, which is expressed on EC but not on Kasumi-1 cells, was used as the primary antibody. Cocultures without primary antibodies in the staining procedure served as the negative controls.

Statistical Analysis
The Student’s t-test, run on Excel 5.0, was used to test for differences between the means of DC output, frequency, and T-cell proliferation in the different groups.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion Conclusion References

 
CD34⁺ Cells in Contact with TNF--Stimulated EC Develop into Dendritic Cells

Morphology   As observed in cytospin preparations (Fig. 1A–1C), CD34⁺ cells in direct contact with unstimulated EC remained in an undifferentiated mononuclear precursor state (Fig. 1A), whereas those in direct contact with TNF--stimulated EC developed typical dendrites (Fig. 1B). When IL-1ß or IL-4 was used instead of TNF-, the cells did not develop a DC-like morphology (not shown). CD34⁺ cells grown in a cytokine cocktail without endothelium became "hairy" and nearly twice as large as those generated on TNF--stimulated EC (Fig. 1C).

View larger version (81K): [in this window] [in a new window]   Figure 1. Morphology of CD34+-derived dendritic cells after 15 days. A) CD34+ cells on unstimulated EC. The cells retained an undifferentiated progenitor morphology. B) DC generated on TNF--stimulated EC developed a typical dendrite morphology. C) DC generated in cytokines only. On average, "hairy" cells were twice as large as those generated in coculture with TNF--stimulated EC (400x magnification).

Immunophenotype   CD34⁺ cells on TNF--stimulated EC also developed the immunophenotype of mature DC (Fig. 2), as demonstrated by the upregulation of CD83 and HLA-DR. These cells were also positive for the costimulatory molecules CD1a, CD80, and CD86 but lacked the monocytic marker CD14. Expression of DC-typical glycoproteins occurred with and without direct contact between EC and developing DC. The peak frequency (26%) of CD83⁺DR⁺ cells in direct contact with TNF--stimulated EC was achieved after 2 weeks.

View larger version (43K): [in this window] [in a new window]   Figure 2. Receptor repertoire of DC developing from CD34+ cells. This figure shows CD83, HLA-DR, CD1a, CD80, and CD86 expression by CD34+ cells cultured in direct contact with (contact) or on a transmembrane above TNF--stimulated EC (noncontact) for 15 days. The numbers in each box represent the frequency of positive cells. One representative result for 11 different experiments is shown.

Other Cytokines   We also investigated the effects of two other inflammatory cytokines, IL-4 and IL-1ß. Although IL-1ß significantly promoted granulocyte expansion in the presence of endothelium, no upregulation of dendritic markers was observed. Stimulation with IL-4 led to the exhaustion of cells in the supernatant after day 23 with no effect on DC differentiation or cell expansion.

Cumulative Generation   The cumulative generation of CD83⁺DR⁺ cells on TNF--stimulated endothelium was followed for 43 days (Fig. 3A). In samples with direct contact to TNF--stimulated EC, the cumulative number of CD83⁺DR⁺ cells generated from 10⁵ CD34⁺ cells after 43 days was 198.6 ± 36.7 x 10³. Without direct contact, the cumulative number was significantly lower (92.3 ± 15.9 x 10³, p < 0.05).

View larger version (23K): [in this window] [in a new window]   Figure 3. A) Cumulative expansion of CD83+DR+ cells derived from CD34+ cells. The culture conditions were as follows: 105 CD34+ cells in direct contact with unstimulated EC (no TNF; circles), in direct contact with TNF--stimulated EC (contact TNF; squares), on a 0.4 µm transmembrane above a monolayer of TNF--stimulated EC monolayer (noncontact TNF; triangles), and in direct contact with TNF-stimulated marrow stroma cells (MSC-TNF; open circles). In the endothelial coculture, each point represents the average of 11 single values except on day 43 (no TNF: n = 5; contact TNF: n = 7; noncontact TNF: n = 3). Dotted line: CD34+ cells in culture medium with TNF- (50 ng/ml) in absence of EC. B) Allogeneic T-cell stimulatory capacity of CD34+ cells cultured on TNF--stimulated endothelium. After 15 days of coculture with unstimulated EC (no TNF; white), in direct contact with TNF--stimulated EC (contact TNF; black), spatially separated from the EC monolayer (noncontact TNF; light gray shaded), and with cytokines only (cytokines; dark gray shaded), the suspended cells were incubated with allogeneic T cells in graded concentrations (average + standard error of one representative experiment in triplicate). DC generated by cytokines only had the highest allostimulatory capacity, followed by DC developed in coculture with TNF--stimulated EC.

Replacement of EC   In the absence of endothelium, the CD34⁺ cells died off by day 6 after the addition of TNF- without significant upregulation of CD83. In samples where EC were replaced by normal marrow stroma cells, about 5% of the CD34⁺ cells expressed CD83 and DR, leading to a cumulative production of around 12.5 ± 10.4 x 10³ dendritic cells. This was regardless of whether the cells were generated in a transmembrane above TNF--stimulated fibroblasts or in direct contact with unstimulated fibroblasts.

Allostimulatory Capacity   As demonstrated by ³H-thymidine incorporation (Fig. 3B), DC from the TNF- coculture systems were capable of inducing allogeneic T-cell proliferation. At the highest concentration of stimulators (5,000; S/R ratio = 1:30), the allostimulatory capacity of CD34⁺-derived DC developing in direct contact with TNF--stimulated EC was similar to that of those without direct contact (19.3 ± 1.9 versus 17.6 ± 2.4 x 10³ cpm; S/R ratio 1:30; p > 0.05). Although the allostimulatory capacity of DC derived on TNF--stimulated EC was lower than that of DC produced by cytokines alone, it was significantly higher than that of progenitor cells cultured on unstimulated EC (11.4 ± 2.1 x 10³ cpm; S/R ratio 1:30; p < 0.05).

AML Cells From Kasumi-1 Cocultured With TNF--Stimulated EC Differentiate Into DC
When cocultured with TNF--stimulated EC, Kasumi-1 cells differentiated into DC and demonstrated the DC immunophenotype within 2 weeks (Fig. 4A). As observed in CD34⁺ cells, the yields of CD83⁺DR⁺ cells were higher in cultures with direct contact to TNF--stimulated EC than in those without direct contact. As shown in Figure 4B, the DC generation dynamics for Kasumi-1 and CD34⁺ cells were similar. The number of CD83⁺DR⁺ cells generated from 10⁵ Kasumi cells was 4.89 ± 0.45 x 10⁵ in cultures with direct contact to TNF--stimulated EC versus 1.48 ± 0.39 x 10⁵ in those without direct contact (noncontact). Cultivation of Kasumi cells with TNF- alone in the absence of EC did not lead to any significant upregulation of CD83. We also investigated the influence of endothelium on the apoptosis rate and cell cycle of developing DC. All Kasumi-1 cells express CD33 and CD45. Since these two markers are not present on endothelium, they allow an immunophenotypic distinction between EC and developing DC. Moreover, due to their tetraploidy, the cell cycle phases of Kasumi-derived DC can be differentiated from those of EC.

View larger version (33K): [in this window] [in a new window]   Figure 4. A) Receptor repertoire of DC developing from leukemic cells. This graph illustrates CD83, HLA-DR, CD1a, CD80, and CD86 expression by Kasumi cells cultured in direct contact with EC (contact) or in a transmembrane above the EC monolayer (noncontact) for 15 days. A high degree of isotype antibody binding was seen in the leukemic cells in direct contact with unstimulated EC. The numbers in each box indicate the frequency of positive cells. B) Cumulative generation of CD83+DR+ cells from leukemic cells. The cumulative DC generation dynamics of DC derived from Kasumi-1 cells were similar to those of CD34+-derived DC. Kasumi cells were cultured on unstimulated EC (no TNF; circles), in direct contact with TNF--stimulated EC (contact TNF; squares), and on a 0.4 µm transmembrane above a monolayer of TNF--stimulated ECs (noncontact TNF; triangles). The averages of three independent experiments are indicated. Dotted line: leukemic cells in culture medium with TNF- (50 ng/ml) in absence of EC.

Apoptosis and Cell Cycle   Developing DC demonstrated a reduction of TNF--induced apoptosis in the presence of endothelium. Under TNF-, the frequency of CD33⁺Annexin⁺ cells decreased from 42% in the absence of endothelium to 23% of DC attached and 16% of DC detached from the EC (p < 0.05, Table 2). The difference between the apoptosis rates in the detached and attached developing DC was not significant (p = 0.06). The cell cycle analysis showed that the G₂M fraction of developing DC in cocultures with direct contact to TNF--stimulated EC nearly doubled, while the S and G_1/0 fractions were lower than those observed in cocultures without direct contact and than those in absence of EC (Table 2). In the unstimulated controls cultured without TNF-, few developing cells attached to the EC monolayer.

Direct Contact Between Developing DC and TNF--Stimulated EC Leads to Internalization of the Developing DC With Bidirectional Protein Exchange

View larger version (63K): [in this window] [in a new window]   Figure 5. Electron microscopy of developing DC and endothelial interactions. In scanning electron microscopy (A), the developing DC in direct contact with TNF--stimulated EC showed pseudopods interdigitating with the endothelial monolayer (5,000x magnification). In transmission electron microscopy (B, 2,784x magnification), developing DC engulfed by endothelium could be observed (arrow), while the membranes of both cell types remained intact (C).

View larger version (54K): [in this window] [in a new window]   Figure 6. Confocal microscopy of endothelium and DC developing from Kasumi cells. Both experiments demonstrated the transfer of GFP from one cell type to the other. A) Developing DC labeled with anti-CD45 and GFP-transduced EC (250x magnification). All CD45+ cells fluoresced green. Size bar: 15 µm. B) Endothelial cells labeled with anti-VCAM and GFP-transduced developing DC (630x magnification). One green-fluorescing developing DC attached to endothelium leads to green fluorescence of the whole endothelial cell. Left upper quadrant: red fluorescence; right upper quadrant green fluorescence; left lower quadrant: red and green fluorescence; right lower quadrant: negative control. Size bar: 5 µm.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion Conclusion References

Development of DC From CD34⁺ Cells Cocultured With TNF--Stimulated EC
Inflammatory cytokines such as IL-1ß, IL-4, and TNF- were tested in this study. IL-1ß has been shown to increase the endothelial production of G-CSF and GM-CSF [35] and to upregulate endothelial receptors important for cell adherence [36]. By day 8, IL-1ß had increased the number of expanded cells tenfold in the direct contact group. However, this only induced the generation of granulocytes and not DC. Similarly, IL-4 also did not lead to DC differentiation, though it is known to suppress the development of monocytes [37] and to increase VCAM-1-dependent T-lymphocyte transmigration [38]. Thus, although these two cytokines are known to increase the expression of endothelial cytokines and several surface molecules [39, 40], neither were able to promote the differentiation of CD34⁺ cells into DC. In contrast, TNF- in synergism with endothelium induced cell differentiation towards the DC lineage. In human endothelial cells, TNF- increases GM-CSF and G-CSF secretion [41]. It also promotes the expression of adhesion molecules important for the attachment and trafficking of leukocytes, lymphocytes, and monocytes [42, 43]. Moreover, TNF- has an important direct effect: by rapidly upregulating GM-CSF receptors while simultaneously downregulating M-CSF and G-CSF receptors on developing progenitors [44], TNF- blocks the differentiation of neutrophils and macrophages, thereby promoting the generation of DC [45, 46].

In our study, the number of DC generated was much higher in cases where CD34⁺ cells were cultured in direct contact with TNF--stimulated EC than in those where they were separated from them by a transmembrane. We attributed this to the observed 1.5- to 2-fold increase in cell expansion, and not to a higher frequency of CD83⁺DR⁺ cells. Although Kasumi-1 AML cells can expand independent of cytokine additives, the frequency of CD83⁺DR⁺ cells was also higher when the cells were cultured in direct contact with TNF--stimulated EC. Therefore, direct cell-to-cell contact seems to support both the expansion of DC precursors and the induction of a dendritic phenotype with an increased allostimulatory capacity. However, since dendritic cells generated by a cytokine combination in the absence of EC induced a higher mixed lymphocyte response, the DCs generated by TNF--stimulated endothelium may have a lower antigen presenting capacity than these cytokine-derived DCs.

In two experiments, CD34⁺ cells were cocultured with human marrow stroma cells instead of EC. Despite stimulation with TNF-, which also promotes the production of fibroblastic G-CSF, M-CSF, and GM-CSF [47, 48], few CD1a⁺14^- cells and even fewer (5%) CD83⁺DR⁺ cells developed. Although other studies demonstrated the ability of lymphoid stroma cells to induce the maturation of dendritic cells [49–51], our findings do not confirm the same capacity for marrow stroma cells. This underlines the importance of endothelium in the induction of DC development and further implies that endothelial cells may produce a yet unidentified factor, which, in synergism with TNF-, favors antigen-presenting cell differentiation from oligopotential myeloid progenitors. Randolph et al. came to similar conclusions about monocytes when they demonstrated that peripheral blood monocytes differentiate into DC by transendothelial migration [13].

It is important to note that DC generation from CD34⁺ progenitor cells lasted more than 40 days, whereas in standard cytokine combinations, DC production usually exhausts after 2 weeks [52, 53]. This suggests that TNF--treated EC are capable of providing cell contact-dependent proliferative or survival signals to DC precursors that allow them to remain in the endothelial layer for a prolonged period, thus providing a persistent source of DC.

Intercellular Dialogue
In light of these results, we analyzed the effects of endothelial contact on apoptosis and cell cycle of the developing DC. The apoptosis rate was lower in cocultures with direct contact to TNF--stimulated EC than in those without direct contact to TNF--stimulated EC or in absence of EC. Under in vitro conditions, fully differentiated DC usually undergo rapid apoptosis [29]. This can be delayed by treating them with TNF family members like TNF-related activation-induced cytokine (TRANCE) [54]. In contrast to fibroblasts, TNF--stimulated human endothelium may provide a factor, which, like TRANCE, improves DC survival. Ferrero et al. came to the same conclusion about monocytes [55].

With the help of electron microscopy, we observed how one developing DC was partially internalized by TNF--stimulated endothelium, while the membranes of both cell types remained intact. This might represent emperipoiesis, a form of transmigration of one cell type through another [56], as has been reported to occur in T cells and epithelium [57]. Endothelial cells probably function as a shelter for developing DC. In doing so, they not only reduce the rate of apoptosis but also induce the proliferation of DC.

In vitro, the yet immature DC attach to the culture bottom, then detach and become quiescent as they mature [2, 58, 59]. This explains why our coculture system had a low G₂M fraction of developed (mature) DC that floated detached in the supernatant above the TNF--stimulated EC monolayer. However, the G₂M fraction of developing DC in direct contact with TNF--stimulated EC was almost twice as high as that of those in indirect contact with TNF--stimulated EC or in absence of EC. Ligand-receptor interactions between endothelium and developing DC is one possible explanation. Furthermore, direct contact between EC and developing DC might induce the secretion of further survival factors that prevent the exhaustion of DC precursors.

A bidirectional transfer of proteins between developing DC and EC was observed by confocal microscopy using GFP as the marker protein. This protein exchange may account for the observed decrease in TNF--dependent apoptosis and may also promote cell proliferation. Eissner et al. described a similar bidirectional cross-talk between endothelium and monocytes/macrophages that reduced the secretion of soluble death factors [60].

One limitation of our study was the isolated use of only three inflammatory cytokines. When an inflammation occurs in vivo, a cytokinetic "fireworks" naturally results in an orchestrated activation of cellular defense mechanisms [61]. IL-1ß in concert with TNF- could induce the proliferation of hematopoietic progenitors by expanding the DC progenitor population before they develop into DC. Interferons and CD40 ligand, which also play a role in DC maturation [4, 62, 63], could further enhance TNF- and endothelial cells in the recruitment and differentiation of DC precursors.

	CONCLUSION

Top Abstract Introduction Materials and Methods Results Discussion Conclusion References

 
Activated human EC, in contrast to marrow stroma cells, can induce DC development from CD34⁺ and leukemic cells. While IL-1ß and direct endothelial contact support progenitor expansion, TNF- is crucial for DC development. Direct contact with the endothelium protects the developing DC from apoptosis, induces cell cycling, and involves a bidirectional exchange of proteins between EC and developing DC. In addition to supplying the ideal combination of cytokines, endothelium also provides cytoadhesion molecules that specifically enhance the attachment, differentiation, survival, and functional capacities of the DC generated. The isolation of the endothelial differentiation factor, which is stimulated by TNF- and induces DC differentiation, as well as the identification of the cellular receptors responsible for the survival of DC precursors, remain objectives for future studies.

	REFERENCES

Top Abstract Introduction Materials and Methods Results Discussion Conclusion References

 

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