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*
Department of Medicine and Vanderbilt Cancer Center, Vanderbilt University Medical Center, Nashville, TN 37232;
Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA 15213; and
Department of Pathology and Cardinal Bernardin Cancer Center, Loyola University Medical Center, Maywood, IL 60153
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B. These data suggest that VEGF interferes with the ability of FL
to promote dendritic cell differentiation from bone marrow progenitor
cells in mice and therefore may decrease the therapeutic efficacy of FL
in settings where increased numbers of DCs might provide clinical
benefits. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tumor cells appear to have developed mechanisms to avoid immune system recognition and elimination, and among these is the inhibition of DC generation and function. Defective function of DCs in cancer has been recently reported by several groups (11, 12, 13, 14, 15, 16). Previously, we demonstrated that one of the possible mechanisms of DC dysfunction in cancer is an abnormal functional maturation of these cells from hemopoietic progenitors (17, 18, 19). Others recently reported similar results (20, 21, 22). Several soluble factors have been implicated in defective DC maturation in cancer, including vascular endothelial growth factor (VEGF).
VEGF is produced in large amounts by most tumors and its production is
closely associated with a poor prognosis (23, 24). VEGF
stimulates the proliferation of endothelial cells and plays an
important role in the formation of tumor neovasculature (reviewed in
Ref. 25). We have demonstrated that anti-VEGF
neutralizing Ab blocks the inhibitory effects of tumor cell
supernatants on DC maturation in vitro (18). A direct
association between increased levels of VEGF in tumor cells and low
presence of DCs in the vicinity of the tumor has been described in 140
patients with gastric cancer (26). VEGF binds to
CD34+ hemopoietic progenitor cells (HPC) through
one of the VEGF-specific receptors, FLT1, and inhibits the activation
of transcription factor NF-B in these cells (27).
Continuous VEGF infusion, at rates as low as 50 ng/h, resulted in a
dramatic inhibition of DC development, associated with an increase in
the production of B cells and immature Gr-1+
myeloid cells (28). Infusion of VEGF was also associated
with inhibition of the activity of the transcription factor NF-B in
bone marrow progenitor cells (28). These data suggest that
VEGF, at pathologically relevant concentrations in vivo, may exert
effects on pluripotent stem cells that results in blocked DC
development. We hypothesize that VEGF might interfere with FL effects
on hemopoietic precursors and hence could be responsible for the
decreased efficacy of FL observed in established tumor models.
Investigation of VEGF/FL interactions might also help to clarify the
mechanisms of action of these two growth factors on hemopoiesis. In
this study, we demonstrate that VEGF partially blocks the ability of FL
to stimulate DC production and maturation in mice in vivo. It appears
that VEGF and FL may act on the same HPC. VEGF does not affect the
expression of FLT3, the receptor for FL, on these cells, but rather may
act through the inhibition of transcription factor NF-B. These
findings suggest that FL-based immunotherapy of cancer might be
enhanced by the co-administration of agents that can block the
inhibitory effects of tumor-derived factors like VEGF.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Six- to 8-wk-old female BALB/c, C57BL/6, and CBA mice were purchased from Harlan (Indianapolis, IN) and were housed in specific pathogen-free units of the Division of Animal Care at Vanderbilt University Medical Center.
Abs and reagents
VEGF was a generous gift from Genentech (South San Francisco,
CA). FL was a generous gift from Immunex (Seattle, WA). The following
Ab-producing hybridomas were obtained from American Type Culture
Collection (Manassas, VA) and used as culture supernatants:
anti-CD4 (L3T4, TIB-207), anti-CD8 (Lyt-2.2, TIB-210),
anti-MHC class II (TIB-120). Mouse GM-CSF, IL-4, TNF-
, and
VEGF165 were obtained from R&D Systems
(Minneapolis, MN); polyclonal anti-mouse Ig was obtained from Sigma
(St. Louis, MO). FITC- or PE-conjugated anti-CD11c, CD11b, CD86
(B7-2), IAd Abs were purchased from PharMingen
(San Diego, CA). Isotype-matched FITC- and PE-conjugated IgG were used
as a control of nonspecific binding. In some experiments, complete
culture medium was used: RPMI 1640 (Life Technologies, Gaithersburg,
MD) with 100 IU/ml penicillin, 0.1 mg/ml streptomycin, 5 x
10-5 M 2-ME, and 10% heat-inactivated FCS
(HyClone, Logan, UT).
VEGF and FL administration
VEGF was delivered into BALB/c mice via Alzet osmotic pumps (Alza, Palo Alto, CA) for 14 days at a rate of infusion of 100 ng/h. The pumps were inserted s.c. into the back of the mice through a small skin incision. Wound edges were reapproximated with surgical clips. All procedures were performed in aseptic conditions and were approved by the Vanderbilt University Animal Care Committee. In controls, pumps were filled with PBS. On day 4 after initiation of VEGF infusion, administration of FL was started. FL was injected s.c. at a dose of 10 µg per mouse daily for 10 days.
Cell separation and analysis of cell-surface receptors
A single-cell suspension was prepared from inguinal, axillary, and brachial lymph nodes, spleens, and thymus by pressing the tissues through a wire mesh. Red cells were removed by hypotonic shock. No additional purification was performed. Cells were washed and used in additional experiments. For analysis of cell-surface receptors, cells were washed in PBS supplemented with 0.1% FCS and labeled with appropriate Abs for 30 min at 4°C. Cells were then washed and analyzed on FACScan flow cytometer (Becton Dickinson, Mountain View, CA). An enriched T cell population was obtained from the lymph nodes by removing adherent cells followed by 1 h incubation on plastic. Bone marrow was obtained from the femurs and tibias of BALB/c mice. In some experiments, marrow cells were cultured for 7 days with GM-CSF and IL-4 in complete medium to generate DCs as described elsewhere (17). In some experiments, bone marrow cells were enriched for HPC by depletion of lineage-specific cells via incubation with the mixture of Abs (TIB-207, TIB-210, TIB-120, and anti-mouse Ig) and complement (low-tox guinea pig complement; Cedarline, Hornby, Ontario, Canada) and subsequent gradient centrifugation on Lympholyte M (Cedarline) to remove dead cells.
Immunohistochemical analysis
Tissues from control and treated animals were snap-frozen in OCT compound and stored at -80°C. Then, 6-µm tissue sections were air-dried and fixed in acetone, rehydrated in PBS, and blocked for nonspecific staining with 2% normal goat serum in PBS. Primary anti-mouse CD11c, CD86 (PharMingen), or NLDC-145 (DEC 205) (Serotec, Oxford, England) Abs were applied for 60 min at room temperature. All slides were subsequently washed in PBS and incubated 30 min with biotinylated secondary Abs (Jackson ImmunoResearch, West Grove, PA). Bound Abs were detected with ABC reagent and Vectastain substrate kit (Vector Laboratories, Burlingame, CA). For the hematoxylin and eosin staining, murine tissues were fixed in 10% neutral buffered formalin and embedded in paraffin. Then, 4-µm sections were cut and stained with hematoxylin and eosin.
Assessment of colony formation
Colony formation by HPC was measured using semisolid 1% methylcellulose medium supplemented with recombinant cytokines (Epo, stem cell factor, IL-6, IL-3) supporting the optimal growth of burst forming unit (BFU)-erythrocyte (E), CFU-granulocyte-macrophage (GM), CFU-macrophage (M), CFU-granulocyte (G), and CFU-mixed erythrocyte-granulocyte-macrophage (GEMM) colonies (Methocult M3434; Stemcell Technologies, Vancouver, Canada). Bone marrow cells were seeded at 15,000 cells per plate. BFU-E colonies were scored on day 8–9 and all other colonies were enumerated on day 12–13.
T cell proliferation assay
Lymph node and spleen cells obtained from control, VEGF-, and FL-treated mice were irradiated (20 Gy), plated in triplicate, and incubated with enriched T cells obtained from CBA mice for a 4-day allogeneic MLR. Twenty-four hours before harvesting, all cultures were pulsed with 1 µCi of [3H]thymidine (Amersham, Arlington Heights, IL). Incorporation of [3H]thymidine was counted using a liquid scintillation counter and expressed as cpm.
EMSA
Double-stranded oligonucleotide probes were prepared by
annealing the appropriate single-stranded oligonucleotides at 65°C
for 10 min in 10 mM Tris, 1 mM EDTA, 10 mM NaCl solution followed by
slow cooling to room temperature. The probes were end-labeled with
[32P]-labeled CTP by filling in 5' overhangs
with the Klenow fragment. The following murine intronic
-chain B site probe was used in this study: wild-type,
5'-AGTTGAGGGGACTTTCCCAGG; mutant B site,
5'-AGTTGAGGCGACTTTCCCAGG.
Nuclear extract was obtained from the cells as described (29). Next, 10 µg of nuclear extract was incubated for 20 min with labeled probe (50,000 cpm) in the presence of 4 µg of poly(dI-dC) (Pharmacia) in binding buffer (20 mM HEPES, 5% glycerol, 0.2 mM EDTA, 1 mM DTT, 5 mM MgCl2). Competition assays were performed with a 100-fold excess of unlabeled probes. The DNA-protein complexes were separated on 4% polyacrylamide gels and visualized and analyzed on a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
RNA preparation and analysis of gene expression
A total of 3 x 106 bone marrow progenitor cells were cultured in complete culture medium in the presence or absence of GM-CSF, VEGF, and FL at 37°C for a different time. RNA was then extracted using the GlassMAX RNA microisolation spin cartridge system (Life Technologies). RT-PCR was performed on RNA samples following a DNA digestion. Reverse transcription was performed using SuperScript preamplification system for first-strand cDNA synthesis (Life Technologies). Briefly, 1 µg of RNA was incubated with 100 ng random hexamers for 10 min at 70°C. Then, 14 µl of the reaction mixture (10x PCR buffer, 25 mM MgCl2, 10 mM dNTP mix, 0.1 M DTT) were added to each sample followed by incubation at 25°C for 5 min. Next, 2 µl (400 U) of SuperScript II RT was added to each sample and incubated at 25°C for 10 min. Samples were then incubated at 42°C for 50 min. The following selected oligonucleotide PCR primer pairs for the FLT3 were used: forward primer, 5'-GTGACTGGCCCCCTGGATAACGAG-3'; reverse primer, 5'-TCCAAGGGCGGGTGTAACTGAACT-3'.
Hypoxanthine phosphoribosyltransferase (HRPT)-specific primers were used as a control and have been described elsewhere (17). Samples were denatured for 30 s at 94°C, annealed for 30 s at 53°C, and extended for 45 s at 72°C for 20 cycles of amplification. This number of cycles was selected to avoid saturation of PCR products and was determined after preliminary experiments. Products were visualized by staining with ethidium bromide after electrophoresis in 1% agarose gel. The PCR product sizes for flt3 and hprt were 509 bp and 164 bp, respectively. PCR products were transferred overnight in an alkaline transfer buffer (0.4 N NaOH, 1 M NaCl) onto Hybond N+ nylon transfer membranes (Amersham, Highland Park, IL), hybridized for 2 h in a rapid hybridization buffer (Amersham), and probed with [32P]-labeled oligonucleotide probes: FLT3, 5'-GCTGGGGGCATGCACACTGTCA-3'; HPRT, 5'-GTTGTTGGATATGCCTTGAC-3'. Membranes were then analyzed, and the expression of flt3 mRNA was quantified by Phosphoimager (Molecular Dynamics).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Effect of VEGF infusion on the ability of FL to stimulate DC production in vivo
Two weeks of VEGF infusions did not result in significant changes in the total number of either lymph node or spleen cells. FL increased the total number of lymph node cells almost 2-fold (p = 0.015) and spleen cells almost 8-fold (p < 0.001). Combined administration of VEGF and FL did not significantly alter FL-induced increases in spleen or lymph node cell number (data not shown). Thus, these results demonstrated that VEGF did not abrogate FL-mediated elevation of cell numbers in murine spleen or lymph nodes.
To investigate the effect of VEGF on FL-induced DC generation, lymph
node cells, splenocytes, and thymocytes were labeled with
anti-CD11c, anti-CD11b, and anti-CD86 Abs. FACScan analysis
revealed that VEGF alone did not alter the number of DC in lymph nodes.
FL dramatically increased the percentage of
CD11c+ DC, but not
CD11c-CD11b+ macrophages
in the lymph node (Fig. 1
). The
percentage of CD11c+CD86+
lymph node cells, representing a subpopulation of mature DC, was
increased up to 4-fold in mice treated with FL
(p < 0.01). As shown in Fig. 1, the ability of
FL to increase accumulation of CD11c+ DC in lymph
nodes was significantly decreased by the coadministration of VEGF. The
percentage of CD11c+,
CD11c+CD11b-, and
CD11c+CD86+ DC was 
50%
lower in mice treated with the FL/VEGF combination in comparison with
mice treated with FL alone (p < 0.01). A
similar, although less pronounced, effect of VEGF was observed for the
population of CD11c+CD11b+
cells (p < 0.05) (Fig. 1). Similar results
were obtained after staining of the lymph node tissue with
anti-NLDC-145 Ab (Fig. 2). Treatment
with FL increased the presence of NLDC-145+ DC in
lymph nodes (Fig. 2B). This effect was dramatically reduced
by coadministration of FL with VEGF (Fig. 2D). Thus, VEGF
significantly decreased FL-induced accumulation of DC in murine lymph
nodes in vivo.
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). As expected,
FL dramatically elevated the presence of total
CD11c+ DC in the spleen
(p < 0.001). This increase was mostly due to
the increased proportion of
CD11c+CD11b- DC. A smaller
increase was observed in the population of
CD11c+CD11b+ cells, and
almost no changes were found in the population of
CD11c-CD11b+ macrophages
(Fig. 1). FL did not affect the presence of the
CD11c+CD86+ population of
DC. VEGF slightly decreased the proportion of these FL-expanded cell
populations. However, this effect did not reach statistical
significance (p = 0.05) (Fig. 1). Similar
effects were observed by immunohistological analysis. The dramatic
increase in NLDC-145+ DC after FL administration
was slightly reduced if FL was delivered together with VEGF (Fig. 2, H vs F). Because our previous experiments have shown that 4-wk VEGF infusion induced a significant increase in the proportion of Gr-1+ cells, we investigated whether FL would affect the number of Gr-1+ cells in the spleen. In four independent experiments, FL alone did not change the presence of Gr-1+ cells in murine spleens. Two weeks of VEGF infusion resulted in slightly elevated levels of these cells (18.6 ± 3.6% vs 11.4 ± 3.2% in control, p > 0.05). Interestingly, the combined administration of VEGF and FL induced a more substantial increase in the proportion of splenic Gr-1+ cells (23.3 ± 3.6%; vs 11.4 ± 3.2% in control, p < 0.05). Thus, our data demonstrated that treatment of mice with FL caused DC accumulation in the spleen, which was not abrogated by the coadministration of VEGF.
Next, we studied the effect of FL and/or VEGF on thymic DC.
Infusion of VEGF alone resulted in a significant decrease in the
presence of a population of mature
CD11c+CD86+ DC in the
thymus (p < 0.05). FL administration increased
primarily the proportion of
CD11c+CD8- DC up to
6-fold, whereas the population of
CD11c+CD8+ DC was
increased only 1.5-fold. VEGF almost completely abrogated these effects
of FL on DC accumulation in the thymus (Fig. 1).
Effect of FL and VEGF on the Ag-presenting ability of lymph node cells and splenocytes
We studied whether the alterations in the presence of DC in
tissues following the FL and/or VEGF therapy would be accompanied by
changes of the ability of lymph node and spleen cells to stimulate
allogeneic T cells. Lymph node and spleen cells were isolated from
control and treated mice, irradiated, and used as stimulators in
allogeneic MLR. Analysis of T cell proliferation revealed that VEGF
alone did not affect the ability of lymph node cells to stimulate
allogeneic T cells. However, VEGF significantly inhibited the
immunostimulatory activity of mixed spleen populations
(p < 0.05) (Fig. 3). In contrast, FL dramatically
increased the stimulatory capacity of both spleen and lymph node cells
(p < 0.01). The effect of VEGF on FL activity
was different in lymph node and spleen. VEGF significantly reduced the
stimulatory effect of FL on lymph node, but not spleen, allostimulatory
activity (p < 0.05), but did not change the
ability of FL to increase the activity of spleen APC
(p > 0.05) (Fig. 3). These data are consistent
with the selective inhibitory effects that VEGF had on DC numbers in
the lymph node relative to spleen.
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In the next series of experiments, we examined the effects of VEGF
treatment on the derivation of specific cell populations from
hemopoietic progenitors. First, bone marrow cells were cultured in
GM-CSF and IL-4-supplemented medium, and the presence of different
populations of DC and their functional activity were compared between
the four experimental groups. No difference between the groups was
found in the total number of cells generated under these conditions.
About 60% of cells expressed DC marker CD11c. Infusion of VEGF alone
did not affect the proportion of any of the above described DC
populations. In contrast, administration of FL stimulated the
generation of mature
CD11c+CD86+ DC. The
percentage of these cells increased up to 2-fold in comparison with
control values (p < 0.05). Infusion of VEGF in
FL-treated mice completely abrogated this effect of FL (Fig. 4A).
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B). It is interesting to note that the infusion of VEGF
alone resulted in a slight but statistically significant decrease in
the stimulatory activity of cultured bone marrow-derived DC
(p < 0.05) (Fig. 4B).
To determine whether treatment with FL, VEGF, or their combination
would regulate the accumulation of colony-forming progenitor cells,
bone marrow cells were isolated from control and treated mice. Bone
marrow cells were then cultured in semisolid methylcellulose medium
supplemented with factors supporting the growth of myeloid and
erythroid colonies. In agreement with previously published observations
(28), VEGF infusion resulted in a moderate increase in
number of CFU-GM and CFU-GEMM colonies (p <
0.05) (Fig. 4C). FL treatment also moderately increased the
number of mixed (CFU-GEMM) colonies, and FL dramatically increased the
number of macrophage (10-fold) and granulocyte-macrophage (3-fold)
colonies (p < 0.01) (Fig. 4C). VEGF
infusion almost completely abrogated these stimulatory effects of FL on
myelopoiesis (Fig. 4C). No significant effect on BFU-E was
determined. These data confirmed previous findings that the
administration of FL in mice significantly activates myelopoiesis,
which in turn resulted in accumulation of a large number of DC in
various lymphoid and nonlymphoid tissues (2, 30).
Coadministration of VEGF decreased FL-induced DC expansion in certain
tissues, which was a possible consequence of VEGF’s inhibitory effects
on myelopoiesis.
Mechanisms of VEGF-FL interaction
The data described above indicate that FL and VEGF may act
at similar stages of hemopoiesis and, perhaps, upon the same precursor
cell population. We tested this hypothesis by first determining whether
VEGF down-regulates the expression of FLT3, a receptor for FL, on the
surface of HPC, thus causing these cells to be less responsive to FL.
Bone marrow HPC were cultured in vitro with 3 ng/ml recombinant murine
GM-CSF with or without of 100 ng/ml VEGF, 100 ng/ml FL, or with the
combination of these factors. Our pilot experiments demonstrated that
these concentrations of FL and VEGF provided an optimal functional
effect in vitro (data not shown). Cultured cells were collected after
different period of time, RNA was extracted, and RT-PCR and Southern
blot analysis were performed. Expression of flt3 mRNA was
normalized to the level of the housekeeper gene hprt in each
sample. The results of three independent experiments showed no
discernable effect of VEGF on the level of flt3 mRNA at any
chosen time point or at any factor combination (the result of one of
the experiment is shown in Fig. 5).
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inducible
activation of NF-B in HPC, resulting in inhibition of DC
differentiation (27). Here, we examined whether the same
mechanism could be involved in the VEGF-mediated inhibition of FL
activity. HPC were isolated from control bone marrow cells and treated
with either FL or FL plus VEGF. Nuclear extracts were prepared and EMSA
was performed as described in Materials and Methods.
Analysis of these data revealed that FL induced a high level of NF-B
nuclear translocation and specific DNA binding. The presence of VEGF in
the same cultures significantly inhibited this effect of FL (Fig. 6). These data support the notion that
FL-mediated activation of NF-B in hemopoietic precursors stimulates
their differentiation into functionally active mature DC, which migrate
to and accumulate in a variety of lymphoid and nonlymphoid tissues.
VEGF may decrease this stimulatory effect of FL by inhibiting
activation of transcription factor NF-B in the same precursor cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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+ cells
represent the majority of DC in the thymus (30).
Interestingly, although FL moderately increased the proportion of these
cells, a more profound effect was seen in population of
CD11c+CD8-. The
increase in the proportion of DC in the spleen and lymph node was
accompanied, as expected, by increased allostimulatory capacity of the
total cell populations from these two organs, a function attributed
specifically to DC. FL also stimulated the production of mature DC in
the lymph node based upon a 3-fold increase in the proportion of cells
expressing high levels of the costimulatory molecule B7-2. However,
this effect was not observed in the spleens of the same animals. This
difference might be explained by the fact that murine spleen is
characterized as an active site of hemopoiesis, and spleen-derived DC
may require additional incubation to reach full maturity
(31, 32, 33). This fact could be demonstrated when we compared
the proportion of CD11c+ DC in the spleen and
lymph node in FL-treated mice and the ability of splenic and lymph node
DC to stimulate T cells in an allogeneic MLR (Figs. 1 and 3). As a
result of FL treatment, CD11c+ cells comprised
almost 20% of the total population of nucleated lymph node cells,
whereas in spleen, the proportion of these cells was >40%. However,
when the same cells were tested for their ability to stimulate an
allogeneic MLR, lymph node DC had a >2-fold higher activity than
splenic DC. Presumably, the splenic microenvironment does not provide
an adequate support for the final steps of DC maturation and
activation. VEGF reduced FL effects on DC generation in the lymph node by almost 50%. This was demonstrated by the analysis of DC phenotype and their ability to stimulate T cell proliferation in an allogeneic MLR. Similar effects of FL and VEGF were observed when thymic DC were analyzed. However, VEGF did not significantly change the FL effects in the spleen. The mechanism of this difference is not clear. An explanation may lie in the immature status of splenic DC. As mentioned above, our previous data demonstrated that 4 but not 2 wk of VEGF infusion drove cell differentiation toward Gr-1+ cells. Two weeks of exposure to VEGF only slightly elevated the level of Gr-1+ cells in spleen. The lack of a marked effect of VEGF after 2 wk of treatment could be explained by the rate of DC turnover and necessity to replenish the resident DC. However, if VEGF was administrated together with FL, it resulted in a significant increase in the proportion of Gr-1+ cells. A possible explanation is that FL induced the production of a large number of progenitor cells. VEGF was able to exert its effects on this amplified population, thus enabling the changes in cell populations to become more pronounced.
To study the possible mechanisms of interaction between FL and VEGF, we
determined whether both factors affected hemopoietic progenitor cells.
Bone marrow cells obtained from mice treated with VEGF and FL were
cultured with GM-CSF and IL-4, a cytokine combination that supports the
generation of DC in vitro. The majority of these cells do not express
the CD86 (B7-2) marker. To achieve their terminal maturation stage,
cells need to be activated additionally for 2–3 days with TNF-,
LPS, or CD40 ligand. In this study, we did not induce the final cell
maturation to evaluate the effect of FL on this process. Neither VEGF,
nor FL treatment in mice significantly changed the total number of DC
generated under these conditions. However, FL substantially (almost
2-fold) increased the proportion of
CD11c+B7-2+ cells
(p < 0.05). This effect was associated with a
significant (2- to 3-fold) increase in the stimulatory activity of
these cells in an allogeneic MLR. However, VEGF dramatically reduced
this effect of FL as was seen by the inhibition of both the expression
of B7-2 and the APC activity. It is important to note that the
combination of GM-CSF and IL-4 does not expand lymphoid subpopulations
of DC. Although it is difficult to make a direct comparison between
these results and the effects seen in mature DC populations in vivo,
they demonstrate a similar trend in the effects of VEGF on FL-mediated
expansion of DCs.
These data suggest that VEGF regulated FL activity on the level of progenitor cells. To further characterize this effect, the number of progenitor cells was studied in a colony-formation assay. We found that FL significantly increased the number of macrophage and granulocyte-macrophage colonies. VEGF alone only moderately increased CFU-GM, but not CFU-M colonies. These data are in agreement with previously published observation describing VEGF effects in vivo and in vitro (28, 34). However, VEGF abrogated the FL effects on both types of myeloid colonies.
Thus, VEGF interferes with FL at the level of progenitor cells by blocking the ability of FL to increase the number of DC progenitors. To study the mechanism of these effects, we hypothesized that VEGF down-regulated the expression of FL receptor FLT3 on the surface of HPC and hence decreased the response of the cells to FL. However, our results did not support this hypothesis, as VEGF did not affect the expression of flt3 mRNA at any time examined.
Next, we investigated the role of the transcription factor NF-B in
this phenomenon. This family of transcription factors is composed of
five DNA-binding subunits, p65 (RelA), p50, p52, c-Rel, and RelB, which
act as homo- or heterodimers. In the cytoplasm of quiescent cells,
these subunits are associated with inhibitory molecules of the IB
family. Cell activation by various stimuli like TNF-, LPS, IL-1,
CD40L, etc. results in serine phosphorylation and degradation of IB
with subsequent nuclear translocation of NF-B dimers
(35, 36, 37). NF-B, and specifically RelB, plays a critical
role in differentiation of DC in vivo and in vitro (27, 38, 39). Wu et al. have recently shown that RelB knockout mice lack
myeloid, but not lymphoid, DC, confirming different mechanisms of
regulation of both lineages of DC (40). We have previously
demonstrated the inhibition of TNF- inducible NF-B activation by
VEGF in vitro (27) and in vivo in mice treated with VEGF
(28). Inhibition of NF-B activation was observed after
the first week of infusion and preceded any morphological changes
(28). To date, there is no information available on
whether FL is able to induce NF-B activation. To study this issue,
we treated bone marrow HPC in vitro with FL and assessed NF-B
nuclear translocation and DNA binding using EMSA. We found that FL
markedly induced NF-B activation. The addition of VEGF dramatically
reduced this effect. Thus, VEGF was able to block NF-B activation.
The consequences of this inhibition are not known yet. It is plausible
to suggest that it might lead to a differential expression of genes
responsible for DC differentiation. The genes possibly involved in this
process are currently under investigation.
In conclusion, this study for the first time demonstrates that the FL effect on DC generation can be inhibited by VEGF. DC play a central role in induction of antitumor immune responses. Therefore, increased production of functionally active DC might be beneficial for cancer treatment. This study demonstrates that tumor-derived factors like VEGF might negatively affect the therapeutic efficacy of FL-based immunotherapies. VEGF inhibition might thus improve the efficiency of FL treatment. In support of this concept, we recently demonstrated that DC function is enhanced in tumor-bearing mice following administration of anti-VEGF Ab (19).4
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Dmitry I. Gabrilovich, Cardinal Bernardin Cancer Center, Loyola University Medical Center, Building 112, Room 203, Maywood, IL 60153. E-mail address:
3 Abbreviations used in this paper: DC, dendritic cell; FL, FLT3 ligand; VEGF, vascular endothelial growth factor; HPC, hemopoietic progenitor cells; BFU, burst-forming unit; E, erythrocyte; G, granulocyte; M, macrophage; GM, granulocyte-macrophage; GEMM, mixed granulocyte-erythrocyte-macrophage.
4 D. I. Gabrilovich, T. Ishida, S. Nadaf, J. E. Ohm, and D. P. Carbone. Antibodies to vascular endothelial growth factor enhance the efficacy of cancer immunotherapy by improving endogenous dendritic cell function. Submitted.
Received for publication March 15, 1999. Accepted for publication July 9, 1999.
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References |
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B activation in hematopoietic progenitor cells. J. Immunol. 160:1224.B. Proc. Natl. Acad. Sci. USA 86:2336.B/rel transcription factor and IB inhibitor system. Curr. Opin. Cell Biol. 5:477.[Medline]
B/IB family: intimate tales of association and dissociation. Genes Dev. 9:2723.B and rel proteins: evolutionarily conserved mediators of immune responses. Annu. Rev. Immunol. 16:225.[Medline]
B/Rel family. Cell 80:331.[Medline]
(-) dendritic cells but not of lymphoid-related CD8(+) dendritic cells. Immunity 9:839.[Medline]
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I. Fricke, N. Mirza, J. Dupont, C. Lockhart, A. Jackson, J.-H. Lee, J. A. Sosman, and D. I. Gabrilovich Vascular Endothelial Growth Factor-Trap Overcomes Defects in Dendritic Cell Differentiation but Does Not Improve Antigen-Specific Immune Responses Clin. Cancer Res., August 15, 2007; 13(16): 4840 - 4848. [Abstract] [Full Text] [PDF]
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A. C. Ochoa, A. H. Zea, C. Hernandez, and P. C. Rodriguez Arginase, Prostaglandins, and Myeloid-Derived Suppressor Cells in Renal Cell Carcinoma Clin. Cancer Res., January 15, 2007; 13(2): 721s - 726s. [Abstract] [Full Text] [PDF]
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 M. M. Dikov, J. E. Ohm, N. Ray, E. E. Tchekneva, J. Burlison, D. Moghanaki, S. Nadaf, and D. P. Carbone Differential Roles of Vascular Endothelial Growth Factor Receptors 1 and 2 in Dendritic Cell Differentiation J. Immunol., January 1, 2005; 174(1): 215 - 222. [Abstract] [Full Text] [PDF]
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 A. Hoeben, B. Landuyt, M. S. Highley, H. Wildiers, A. T. Van Oosterom, and E. A. De Bruijn Vascular Endothelial Growth Factor and Angiogenesis Pharmacol. Rev., December 1, 2004; 56(4): 549 - 580. [Abstract] [Full Text] [PDF]
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 S. M. Stick and P. G. Holt The Airway Epithelium as Immune Modulator: The LARC Ascending Am. J. Respir. Cell Mol. Biol., June 1, 2003; 28(6): 641 - 644. [Full Text] [PDF]
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 D. I. Gabrilovich, P. Cheng, Y. Fan, B. Yu, E. Nikitina, A. Sirotkin, M. Shurin, T. Oyama, Y. Adachi, S. Nadaf, et al. H1{degrees} histone and differentiation of dendritic cells. A molecular target for tumor-derived factors J. Leukoc. Biol., August 1, 2002; 72(2): 285 - 296. [Abstract] [Full Text] [PDF]
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 T. Teshima, P. Reddy, K. P. Lowler, M. A. KuKuruga, C. Liu, K. R. Cooke, and J. L. M. Ferrara Flt3 ligand therapy for recipients of allogeneic bone marrow transplants expands host CD8alpha + dendritic cells and reduces experimental acute graft-versus-host disease Blood, March 1, 2002; 99(5): 1825 - 1832. [Abstract] [Full Text] [PDF]
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Z. Dembic, J.-A. Rottingen, J. Dellacasagrande, K. Schenck, and B. Bogen Phagocytic dendritic cells from myelomas activate tumor-specific T cells at a single cell level Blood, May 1, 2001; 97(9): 2808 - 2814. [Abstract] [Full Text] [PDF]
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 M. A. Morse, S. Nair, M. Fernandez-Casal, Y. Deng, M. St Peter, R. Williams, A. Hobeika, P. Mosca, T. Clay, R. I. Cumming, et al. Preoperative Mobilization of Circulating Dendritic Cells by Flt3 Ligand Administration to Patients With Metastatic Colon Cancer J. Clin. Oncol., December 1, 2000; 18(23): 3883 - 3893. [Abstract] [Full Text] [PDF]
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