Tumor Necrosis Factor Alpha-Stimulated Endothelium: An Inducer of Dendritic Cell Development from Hematopoietic Progenitors and Myeloid Leuk
http://www.100md.com
《干细胞学杂志》
a Institute for Transfusion Medicine, Charité, Universit?tsmedizen 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, Universit?tsmedizin Berlin, Institute for Transfusion Medicine, Augustenburger Platz 1, 13353 Berlin, Germany. Telephone: 49-160-1090837; Fax: 49-30-450-553988; e-mail: anja.moldenhauer@charite.de
ABSTRACT
Dendritic cells (DC), the most potent antigen-presenting cells, serve as a nexus between immunogenicity and tolerogenicity . 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 , and B) differentiation of CD34+ hematopoietic progenitors and leukemic cells into DC . While monocytes can differentiate into DC within 48 hours , CD34+ cells usually need 7–12 days .
Endothelial cells (EC) have been demonstrated to recruit granulocytes , lymphocytes , and monocytes , the latter even differentiate into DC while transmigrating through endothelium . Furthermore, EC also support the proliferation of CD34+ cells by constitutive production of cytokines and direct cellular contact . 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 . Besides, CD34+-derived DC have been shown to be less allostimulatory than monocyte-derived DC .
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 . 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 .
We therefore hypothesized that the presence of inflamed endothelium simulated by stimulating a monolayer of EC with tumor necrosis factor (TNF)-, IL-1?, 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
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-1? 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).
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.
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.
Table 1 demonstrates the total number of cells harvested per week and the frequency of CD83+DR+ cells per sampling point until day 30. In all cocultures with TNF--stimulated EC, the frequency of CD83+ and HLA-DR+ cells was significantly higher, and the total number of cells harvested was twice as high as in those with unstimulated EC (p = 0.01). Compared with the amount harvested in direct contact with the TNF--stimulated EC, spatial separation of the hematopoietic progenitors and stimulated EC by a microporous membrane reduced the total number of DC harvested by half (day 15). Although the frequencies of CD83+HLA-DR+ cells were equivalent, the total numbers of cells harvested from the transmembrane systems were as low as those observed in the cocultures with unstimulated EC (p > 0.05). As a result, the number of DC generated by indirect contact with TNF--stimulated EC was only half as high as that obtained by direct contact on day 15.
Table 1. Cell count and frequency of CD83+DR+ cells derived from CD34+ cells in coculture with endothelium
Other Cytokines ? We also investigated the effects of two other inflammatory cytokines, IL-4 and IL-1?. Although IL-1? 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).
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.
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 G1/0 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.
Table 2. Cell cycle (%) and apoptosis rate (%) of DC developing from Kasumi cells
Direct Contact Between Developing DC and TNF--Stimulated EC Leads to Internalization of the Developing DC With Bidirectional Protein Exchange
As observed by scanning electron microscopy (Fig. 5A), DC progenitor cells developed cytoplasmic tethering sites that connected them to the EC monolayer within 24 hours of coculture. Transmission electron microscopy (Fig. 5B) demonstrated that one developing DC or one of its dendrites was internalized by a TNF--activated EC, while the membranes of both cells remained intact (Fig. 5C).
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).
To visualize a direct protein transfer between endothelium and developing DC, Kasumi cells were plated on GFP-transduced, TNF--stimulated EC. All CD45+ developing DC attached to the EC monolayer fluoresced green within 24 hours (Fig. 6A). In the reverse experiment, green-fluorescent developing DC transmitted GFP to the VCAM+ EC (Fig. 6B). Following separation, GFP positivity disappeared within 2 days.
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
Activated human EC, in contrast to marrow stroma cells, can induce DC development from CD34+ and leukemic cells. While IL-1? 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.
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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, Universit?tsmedizin Berlin, Institute for Transfusion Medicine, Augustenburger Platz 1, 13353 Berlin, Germany. Telephone: 49-160-1090837; Fax: 49-30-450-553988; e-mail: anja.moldenhauer@charite.de
ABSTRACT
Dendritic cells (DC), the most potent antigen-presenting cells, serve as a nexus between immunogenicity and tolerogenicity . 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 , and B) differentiation of CD34+ hematopoietic progenitors and leukemic cells into DC . While monocytes can differentiate into DC within 48 hours , CD34+ cells usually need 7–12 days .
Endothelial cells (EC) have been demonstrated to recruit granulocytes , lymphocytes , and monocytes , the latter even differentiate into DC while transmigrating through endothelium . Furthermore, EC also support the proliferation of CD34+ cells by constitutive production of cytokines and direct cellular contact . 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 . Besides, CD34+-derived DC have been shown to be less allostimulatory than monocyte-derived DC .
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 . 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 .
We therefore hypothesized that the presence of inflamed endothelium simulated by stimulating a monolayer of EC with tumor necrosis factor (TNF)-, IL-1?, 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
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-1? 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).
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.
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.
Table 1 demonstrates the total number of cells harvested per week and the frequency of CD83+DR+ cells per sampling point until day 30. In all cocultures with TNF--stimulated EC, the frequency of CD83+ and HLA-DR+ cells was significantly higher, and the total number of cells harvested was twice as high as in those with unstimulated EC (p = 0.01). Compared with the amount harvested in direct contact with the TNF--stimulated EC, spatial separation of the hematopoietic progenitors and stimulated EC by a microporous membrane reduced the total number of DC harvested by half (day 15). Although the frequencies of CD83+HLA-DR+ cells were equivalent, the total numbers of cells harvested from the transmembrane systems were as low as those observed in the cocultures with unstimulated EC (p > 0.05). As a result, the number of DC generated by indirect contact with TNF--stimulated EC was only half as high as that obtained by direct contact on day 15.
Table 1. Cell count and frequency of CD83+DR+ cells derived from CD34+ cells in coculture with endothelium
Other Cytokines ? We also investigated the effects of two other inflammatory cytokines, IL-4 and IL-1?. Although IL-1? 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).
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.
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 G1/0 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.
Table 2. Cell cycle (%) and apoptosis rate (%) of DC developing from Kasumi cells
Direct Contact Between Developing DC and TNF--Stimulated EC Leads to Internalization of the Developing DC With Bidirectional Protein Exchange
As observed by scanning electron microscopy (Fig. 5A), DC progenitor cells developed cytoplasmic tethering sites that connected them to the EC monolayer within 24 hours of coculture. Transmission electron microscopy (Fig. 5B) demonstrated that one developing DC or one of its dendrites was internalized by a TNF--activated EC, while the membranes of both cells remained intact (Fig. 5C).
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).
To visualize a direct protein transfer between endothelium and developing DC, Kasumi cells were plated on GFP-transduced, TNF--stimulated EC. All CD45+ developing DC attached to the EC monolayer fluoresced green within 24 hours (Fig. 6A). In the reverse experiment, green-fluorescent developing DC transmitted GFP to the VCAM+ EC (Fig. 6B). Following separation, GFP positivity disappeared within 2 days.
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
Activated human EC, in contrast to marrow stroma cells, can induce DC development from CD34+ and leukemic cells. While IL-1? 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.
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