Published online before print August 30, 2007
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* Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, and Department of Medical Research, National Taiwan University Hospital, Taipei, Taiwan, Republic of China; and
Graduate Institute of Biomedical Technology, College of Medicine, Taipei Medical University, Taipei, Taiwan, Republic of China
1Correspondence: Department of Pediatrics, National Taiwan University Hospital, No. 7, Chung-Shan South Road, Taipei, Taiwan, R.O.C. E-mail: gicmbor{at}ntu.edu.tw
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production in response to LPS stimulation. This PlGF-induced DC dysfunction was recovered by anti-human VEGF receptor 1 mAb. In addition, treatment of PlGF-DC or classical-DC with PlGF resulted in the suppression of naïve CD4+ T cell proliferation in an allogenic MLR but up-regulated the IL-5 and IL-13 secretion of the CD4+ T cell. PlGF was also able to inhibit LPS-induced I
B phosphorylation and NF-B activity. Taken together, our data demonstrate that the immunosuppressive properties of PlGF are through the NF-B signaling pathway. PlGF might play a major role in the pathogenesis of tumors and act as an effector molecule to skew T cell response to the Th2 phenotype, which might be more beneficial for pregnancy.
Key Words: T cells • Th1/Th2 cells • signal transduction
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), bacterial products (e.g., LPS and unmethylated DNA CpG motif), and contact sensitizers, can induce DC maturation in vivo and in vitro [5
, 6
]. Several reports have already indicated that the nuclear transcription factor NF-B also plays an important role in DC maturation [7
]. Persico et al. [8 ] originally discovered the placenta growth factor (PlGF) in human placenta in 1991. PlGF belongs to the vascular endothelial growth factor (VEGF) family and represents a key regulator of angiogenic events in development and pathologic conditions. The VEGF-A homodimer exerts its biological activities through the activation of two distinct tyrosine kinase receptors: fms-like tyrosine kinase receptor-1 (also known as VEGFR-1) and the kinase domain-containing receptor/fetal liver kinase receptor (also known as VEGFR-2). PlGF is secreted as a glycosylated homodimer, which binds and induces autophosphorylation of VEGFR-1 only [9 , 10 ]. Normal placental tissues express higher levels of PlGF relative to VEGF expression levels [11 ]. Many studies have demonstrated that most cancer cells produce PlGF [12 , 13 ]. Presently, the biological role and signaling mechanisms mediating the cellular action of PlGF remain poorly understood.
To clarify the role of PlGF in pregnancy and in cancer, the exact effects of PlGF on human CD14+ monocyte and DCs are yet to be defined. In the present study, we first examined the molecular mechanisms of PlGF on the human monocyte-derived DCs. We showed that PlGF inhibited LPS-induced maturation of DCs with the down-regulation of IL-12 p70, p40, IL-8, and TNF- cytokine secretion as well as inhibited LPS-induced IB phosphorylation (IB-P) and NF-B activity. Moreover, PlGF-treated DCs biased T cell differentiation to the Th2 phenotype in allogenic MLR. Therefore, the PlGF produced by many tumor cells could suppress host anti-tumor immunity directly by altering DC function, and PlGF produced in placental tissues could skew the immune response from Th1 to Th2, which may be more beneficial for pregnancy.
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Monocyte purification and generation of monocyte-derived DCs
DCs were generated from PBMCs, as described previously, with some modification [14
, 15
]. Briefly, PBMCs were obtained from healthy donors by centrifugation with Ficoll-Hypaque (Pharmacia, Uppsala, Sweden), and the light density fraction from the 42.5% to 50% interface was recovered. CD14+ cells were purified by positive selection using anti-CD14+ microbeads in conjunction with the MiniMACS system by following the manufacturer’s instructions (Miltenyi Biotec, Auburn, CA, USA).
The CD14+ cells were cultured at 1 x 106 cells per 1 ml cRPMI in 24-well plates (Costar, Cambridge, MA, USA) with GM-CSF (800 U/ml) and IL-4 (500 U/ml) for 5 days to differentiate into iDCs in the presence of 200 ng/ml PlGF (referred as "PlGF-DC") or the absence of PlGF (referred as "classical-DC"). On Days 3 and 5, the cells were fed with cRPMI medium containing the above cytokines. Where indicated, PlGF was added on Day 5 to study the effect of PlGF on different stages of DC differentiation. DC maturation was induced by the addition of LPS (100 ng/ml) to the culture on Day 5 for 48 h.
Determination of cytokine levels
The IL-12 p70, IL-12 p40, IL-4, IL-5, IL-8, IL-10, IL-13, TNF-, and IFN-
in the culture supernatant from DCs or T cells were assayed with an ELISA kit (R&D Systems), per the manufacturer’s instructions. Their detection sensitivities were 10, 32, 10, 12, 3.5, 10, 32, 5.5, and 8 pg/ml, respectively.
Flow cytometric analysis
DCs were harvested and washed with cold buffer (PBS containing 2% FCS and 0.1% sodium azide). Cells were then incubated in cold buffer. Subsequent stainings with mAb (including CD1a, CD11c, CD14, CD40, CD80, CD83, CD86, HLA-DR, and M-CSFR) or isotype-matched controls were performed for 30 min on ice. Stained cells were then washed twice and resuspended in cold buffer and analyzed with a FACSort cell analyzer (Becton Dickinson, San Jose, CA, USA). More than 1 x 104 cells were analyzed for each sample, and the results were processed by using CellQuest software (Becton Dickinson).
Neutralization experiments
Human DCs were preincubated for 1 h with 20 µg/ml antibody solution of anti-human VEGFR-1 mAb and anti-human VEGFR-2 mAb (R&D Systems). LPS and PlGF were then added for 15 h. The cell culture supernatants were collected and analyzed for IL-12 p70 and IL-12 p40 by ELISA.
Allogeneic MLR
PBMCs were obtained as described above, and naïve CD4+ T cells were purified by a naïve CD4+ T cell isolation kit (Miltenyi Biotec). The allogenic CD4+ T cells obtained were distributed at 2 x 105 cells per well and incubated for 3 or 5 days in the presence of graded numbers of irradiated DCs (3000 rad, 137Cs source). On Day 3, the culture supernatants were harvested for IL-4, IL-5, IL-10, IL-13, and IFN- analysis. On Day 5, tritiated thymidine (1 µCi/well, New England Nuclear, Boston, MA, USA) was added, and the cells were incubated for another 16 h. The cells were harvested on a cell harvester (Packard Instrument Co., Meriden, CT, USA), and the incorporated radioactivity was measured using a β-counter (Packard Instrument Co.).
Western blotting
Total cellular extract was prepared using Gold lysis buffer. Total protein (50 µg) was separated on 10% SDS-polyacrylamide minigels and transferred to Immobilon polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). The membrane was incubated overnight at 4°C with 10% BSA in PBS to block nonspecific Igs and then incubated with anti-IB-P polyclonal (Cell Signaling Technology, Beverly, MA, USA) and anti--tubulin mAb (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
Preparation of nuclear extracts and EMSA
Nuclear and cytoplasmic extracts were prepared as described previously [16
]. At the end of the culture, the cells were suspended in hypotonic buffer A (10 mM HEPES, pH 7.6, 10 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5 mM PMSF) for 10 min on ice and vortexed for 10 s. Nuclei were pelleted by centrifugation at 12,000 g for 20 s. The supernatants containing cytosolic proteins were collected, and pellets containing nuclei were resuspended in buffer C (20 mM HEPES, pH 7.6, 25% glycerol, 0.4 M NaCl, 1 mM EDTA, 1 mM DTT, 0.5 mM PMSE) for 30 min on ice. The supernatants containing nuclear proteins were collected by centrifugation at 12,000 g for 20 min and stored at –70°C.
For EMSA, each 5 µg nuclear extract was mixed with the labeled, double-stranded NF-B oligonucleotide, 5'-AGTTGAGGGGACTTTCCCAGGC-3' and incubated at room temperature for 20 min. The incubation mixture included 1 µg poly(dI-dC) in a binding buffer (25 mM HEPES, pH 7.9, 0.5 mM EDTA, 0.5 mM DTT, 1% Nonidet P-40, 5% glycerol, and 50 mM NaCl). The DNA–protein complex was electrophoresed on 4.5% nondenaturing polyacrylamide gels in 0.5 x 0.0445 M Tris, 0.0445 M borate, 0.001 M EDTA buffer. A double-stranded, mutated oligonucleotide, 5'-AGTTGAGGCGACTTTCCCAGGC-3', was used to examine the specificity of the binding of NF-B to DNA. The specificity of binding was also examined by comparison with the unlabeled oligonucleotide.
Statistical analysis
The Student’s t-test was used to analyze the results, and a P value of less than 0.05 was considered statistically significant.
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, and IL-10 production of DCs, and IL-10 cytokine concentrations in the supernatants of both types of DCs cultured with different doses of PlGF for 48 h. In the PlGF-DC experiment, we demonstrated that LPS-treated DCs enhanced the production of IL-12 p70, IL-12 p40, and IL-10 compared with untreated DCs, whereas a treatment of various concentrations of PlGF in the presence of LPS impaired the production of IL-12 p70, IL-12 p40, and IL-10 significantly in a dose-dependent manner (Fig. 1A
).
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Figure 1. Modulation of cytokine secretion of DC by PlGF. CD14+ monocytes were cultured with GM-CSF and IL-4 for 5 days to differentiate them into iDCs in the presence of 200 ng/ml PlGF (referred as PlGF-DC) or absence of PlGF (referred as classical-DC). (A) PlGF-DC or (B) classical-DC was treated with medium, various concentrations of PlGF, LPS (100 ng/ml) alone, or LPS plus various concentrations of PlGF for 48 h. At the end of the incubation time, IL-12 p70, p40, and IL-10 production was subsequently analyzed by ELISA. (C) PlGF-DC was treated with medium (Cont), PlGF, LPS, or LPS plus PlGF for 48 h, and then the IL-8 and TNF- production was analyzed by ELISA. Data represent the mean ± SE for three determinations. *, P < 0.05, when compared with LPS-treated DCs.
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compared with LPS-stimulated DCs (Fig. 1C)
. Similar results were obtained when LPS plus PlGF decreased the secretion of IL-8 and TNF- compared with LPS-stimulated DCs in a classical-DC experiment (data not shown).
Modulatory effect of PlGF on DC differentiation and maturation
LPS has been described as an inducer of DC activation and maturation. Therefore, we use LPS as a positive control. In this study, we compared the phenocyte of human PlGF-DC treated with medium, PlGF (200 ng/ml), LPS (100 ng/ml) alone, or LPS plus PlGF for 48 h. We found that stimulation of DC with LPS resulted in up-regulation of CD80, CD86, CD83, CD40, and HLA-DR expression, and CD1a was decreased within 48 h, whereas a treatment of 200 ng/ml PlGF with LPS in PlGF-DC impaired the expression of CD80, CD86, CD83, CD40, and HLA-DR, and CD1a was increased compared with LPS-stimulated DC (Fig. 2
). Similar results were obtained when PlGF was added on classical-DC (data not shown). In both types of DC treated with medium, PlGF (200 ng/ml), LPS (100 ng/ml) alone, or LPS plus PlGF for 48 h, there is no CD14 and M-CSFR expression in the four groups (data not shown).
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Figure 2. Modulation of surface marker expression of DC by PlGF. PlGF-DC was treated with medium (Control), PlGF (200 ng/ml), LPS (100 ng/ml) alone, or LPS plus PlGF for 48 h, and then surface markers were analyzed by flow cytometry (dotted line, isotype control; solid line, specific mAb). The values shown in the flow cytometry profiles are the mean fluorescence intensity by CellQuest software (BD Biosciences, San Jose, CA, USA).
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Figure 3. PlGF inhibits the synthesis of IL-12 p70 and IL-12 p40 in LPS-treated human DC through the VEGFR-1. Human classical-DC was preincubated with 20 µg/ml anti-human VEGFR-1 mAb, anti-human VEGFR-2 mAb, and IgG1 antibodies separately for 1 h. Classical-DC was then challenged with LPS and PlGF for 15 h. The cell culture supernatants were collected for IL-12 p70 and IL-12 p40 analysis. Data are represented as mean ± SE.
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Figure 4. The effects of VEGF on IL-12 p70 and IL-12 p40 production in DC. Human classical-DC was treated with medium, PlGF, VEGF, LPS, LPS plus PlGF, or LPS plus VEGF, and then the supernatants were collected for IL-12 p70 and IL-12 p40 analysis. Data are represented as mean ± SE. *, P < 0.05, when compared with LPS-treated DCs.
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Figure 5. Modulatory effects of PlGF on T cell proliferation in MLR. (A) PlGF-DC or (B) classical-DC was treated with medium, PlGF, LPS, or LPS plus PlGF for 48 h. Allogeneic CD4+ T cells were cultured with -irradiated DC in 96-well plates for 5 days at different DC:T ratios, and the T cell proliferation was measured as described in Materials and Methods. Data represent the mean ± SE for three determinations. #, P < 0.05, when compared with untreated DCs; *, P < 0.05, when compared with LPS-treated DCs.
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In addition to its suppressive effect on T cell proliferation, PlGF modulated the secretion of IFN- and IL-5 of naïve CD4+ T cells. We found that PlGF-treated PlGF-DC enhanced T cell secretion of IL-5 in the supernatant compared with untreated PlGF-DC. LPS plus PlGF-treated PlGF-DC also enhanced T cell secretion of IL-5 compared with LPS-treated PlGF-DC (Fig. 6A
, lower panel). In contrast, PlGF-treated PlGF-DC did not affect IFN- secretion significantly (Fig. 6A
, upper panel). The effect of PlGF-treated classical-DC on IFN- and IL-5 secretion from naïve CD4+ T cells was then tested. When T cells were incubated with irradiated DCs at a DC:T ratio of 1:20, we found that PlGF or LPS plus PlGF-treated classical-DC did not affect the secretion of IFN- (Fig. 6B
, upper panel). Consistently, PlGF or LPS plus PlGF-treated classical-DC enhanced T cell secretion of IL-5 significantly compared with untreated or LPS-treated classical-DC, respectively (Fig. 6B
, lower panel).
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Figure 6. Modulatory effects of PlGF on T cell cytokine secretion in MLR. (A) PlGF-DC or (B) classical-DC were treated with medium, PlGF, LPS, or LPS plus PlGF for 48 h. The concentration of IFN-, IL-5, and (C) IL-13 secreted by naïve CD4+ T cells, which were cocultured with -irradiated DC in 96-well plates for 3 days, was determined by ELISA. These data are means ± SE of triplicates and representative of three independent experiments. #, P < 0.05, when compared with untreated DC; *, P < 0.05, when compared with LPS-treated DC.
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and IL-13:IFN- ratios by enhancing IL-5 and IL-13 secretion.
PlGF suppressed IB-P in DCs
NF-B is one molecular family whose activation is associated with DC maturation. NF-B normally binds to IB, which impedes NF-B nuclear translocation from the cytoplasm to the nucleus. Once cells are exposed to inflammatory stimuli, including LPS and TNF-, IB is phosphorylated, leading to IB degradation and nuclear translocation of NF-B.
We examined whether PlGF had any affect on IB-P. The cytoplasmic levels of IB-P protein were examined by Western blot analysis. After 1 h, LPS-treated PlGF-DC induced the phosphorylation of IB significantly. When PlGF was added on PlGF-DC (Fig. 7A
) or classical-DC (Fig. 7B)
, under both conditions, after 1 h from the activation of human DCs with LPS plus various concentrations of PlGF, the IB-P protein was inhibited significantly in a dose-dependent manner when compared with LPS-treated DCs.
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Figure 7. PlGF inhibited IB-P in LPS-stimulated DCs. (A) PlGF-DC or (B) classical-DC was treated with medium, PlGF, LPS, or LPS plus various concentration of PlGF for 1 h. Cytosolic fractions were prepared and analyzed for the content of IB-P protein by Western blotting. This experiment was repeated three times with similar results.
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B activation in DCsB activation. To determine whether PlGF exerted any affect on NF-B activation, we monitored its ability to affect NF-B translocation into the nucleus. PlGF was added on PlGF-DC (Fig. 8A
) or classical-DC (Fig. 8B)
; under both conditions, DCs were cultured in the presence of medium, PlGF, LPS, or LPS plus PlGF for 2 h, and then nuclear extracts were analyzed for NF-B binding by the EMSA. As shown in Figure 8
, the induction of specific NF-B binding with a NF-B site by LPS was markedly inhibited by the coincubation with PlGF. The binding of NF-B was specific and could be blocked by unlabeled, competing NF-B oligonucleotides.
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Figure 8. PlGF inhibited NF-B activation in LPS-stimulated DCs. (A) PlGF-DC or (B) classical-DC was treated with medium, PlGF, LPS, or LPS plus PlGF for 2 h, and then nuclear fractions were prepared and analyzed for NF-B binding activity by EMSA. To assess the specificity of the binding, 100-fold excess of cold NF-B probe or mutant probe was added to the LPS condition. Band intensities were quantified by densitometry. The arbitrary units represent the relative amounts of the radioactivity present in respective bands. This experiment was repeated three times with similar results.
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secretion on DCs. In addition, PlGF-treated DCs reduced T cell proliferation dramatically and enhanced IL-5 and IL-13 secretion by naïve CD4+ T cells. This is also the first study to demonstrate that PlGF was able to inhibit LPS-induced IB-P and NF-B activity on DCs.
DCs, which are the most potent APCs, play an important role in the host’s immunosurveillance against cancer. Many investigators reported impaired function of DCs in cancer-bearing hosts. DCs fail to present tumor-specific antigen [17
] and transfer it to the naïve T cell in patients with advanced stages of cancer [18
]. To investigate further the alteration of DCs induced by PlGF, we established an in vitro model, where LPS induced maturation of monocyte-derived DCs in the presence or absence of PlGF. We found out that when PlGF was added to the culture of PlGF-DC or classical-DC, the stimulation of DCs with LPS plus PlGF decreased the secretion of IL-12 p70, IL-12 p40, IL-8, and TNF- compared with LPS-stimulated DCs.
In immune responses, IL-12 plays a central role as a link between the innate and adaptive immune systems [19
]. Thus, IL-12 induces and promotes NK and T cells to generate IFN- and lytic activity. In addition, IL-12 polarizes the immune system toward a primary Th1 response. It is interesting that we demonstrated that classical-DC cultured in the presence of PlGF and stimulated with LPS plus PlGF had a slight up-regulation of IL-10 when compared with LPS-stimulated DCs. IL-10 is a pleiotropic cytokine produced by DCs, T cells, and macrophages and possesses anti-inflammatory and immunosuppressive properties [20
]. LPS has also been described as a Th1 inducer. In this study, our data demonstrated that IL-5 and IL-13 cytokines were induced in MLR by PlGF-treated or LPS plus PlGF-treated human DCs, which showed that PlGF might cause DCs to skew the immune response toward Th2 development.
Recent reports show that LPS is one potent DC maturation factor that induces NF-B activation and the phosphorylation of IB in monocyte-derived DCs [21
]. We then sought to establish the signal transduction and transcriptional activation mechanisms altered by PlGF signaling in LPS-treated DCs. The transcription factor NF-B is involved in the regulation of many genes responsible for cell activation; immune, inflammatory, and cytokine responses; cell adhesion; and growth control [22
, 23
]. We therefore hypothesized that NF-B might be the transcription factor most responsible for the observed effects of PlGF on DC maturation.
In our experiments, PlGF inhibited the phosphorylation of IB and blocked the ability of NF-B to activate transcription and bind DNA specifically, consistent with our hypothesis that the reduced NF-B signal transduction pathway may mediate the effects of PlGF. The antigen-presenting function in DC, as a result of abnormal differentiation, is an important mechanism of tumor escape from immune control. Therefore, blockade of NF-B activation in DC by PlGF may be a mechanism by which tumor cells can directly down-modulate the ability of the immune system to generate effective anti-tumor immune responses.
In general, the angiogenic process is initiated by growth factors such as basic fibroblast growth factor, VEGF, or PlGF. The process of angiogenesis is also involved in the development and progression of diseases, including tumor growth and metastases and rheumatoid arthritis [24 , 25 ]. Many studies about cancer cell lines and tumor tissues so far indicate that PlGF expression is up-regulated in human gastric adenocarcinoma, renal cell cancer, melanoma, cervical squamous cell carcinoma, meningiomas, and breast cancer [12 , 13 , 26 ]. Parr et al. [26 ] demonstrated that PlGF expression correlates with breast cancer prognosis, as high levels of PlGF were significantly associated with a higher degree of lymph node involvement, poorly differentiated tumors, and an overall poor outlook for the patient. Our data found that PlGF exerts immunosuppressive properties. Therefore, the processes of tumor formation are not only contributed to by PlGF-induced angiogenesis but also by PlGF-related immunosuppression. Therefore, blockade of PlGF signaling using a humanized anti-PlGF mAb, an anti-human VEGFR-1 mAb, or a small molecule inhibiting VEGFR-1 signal transduction may improve the PlGF-induced DC dysfunction in cancer-bearing hosts, in addition to the antiangiogenetic effects against the tumor microenvironment.
The impact of PlGF, not only on DCs but also on multiple hematopoietic progenitors [27 ], highlights the bifunctional nature of PlGF in pathologic as well as normal, physiologic neoangiogenesis. Placental development requires adequate and organized interaction of vascular growth factors and their receptors, including VEGF and PlGF, which acting through the tyrosine kinase receptors, VEGFR-1 and VEGFR-2, have been implicated in bovine placental vascular development. The PlGF gene is highly expressed in placenta at all stages of human gestation.
During the implantation period, a multidirectional cytokine network is necessary, together with the blastocyst-producing cytokines and other factors, as well as the endometrium-synthesizing factors needed for the embryonic development [28
]. Inadequate immune responses and an unbalanced cytokine network may be related to implantation failures, pregnancy losses, and obstetric complications. The Th2 cytokine profile is believed to be favorable for pregnancy in contrast to Th1 cytokines, which mediate cellular immune responses [29
, 30
]. Our report showed that PlGF-treated DCs increased the IL-5:IFN- and IL-13:IFN- ratios by enhancing IL-5 and IL-13 secretion. We then suggested that PlGF might cause DCs to skew the immune response toward Th2 development.
In conclusion, we demonstrated that PlGF could inhibit the significant activation and maturation of human DCs effectively and rapidly by the NF-B pathway. This study suggests that when cultured with PlGF-DC or classical-DC in vitro, PlGF can modulate the expression of surface molecules and cytokine production, reduce T cell proliferation, and enhance IL-5 and IL-13 production of naïve CD4+ T cells. Moreover, PlGF produced by tumor cells could suppress host anti-tumor immunity directly by altering DC function. Last, PlGF produced in placental tissues may help maintain a Th2 cytokine profile, which may be more beneficial for normal fetal development and may thus be partly responsible for reduced birth defects.
Received March 16, 2007; revised July 12, 2007; accepted August 9, 2007.
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B dependent: coordinate regulation of MHC, co-stimulatory molecules and cytokines Int. Immunol. 13,675-683B and p38 mitogen-activated protein kinase pathways J. Leukoc. Biol. 78,533-543B Mol. Pharmacol. 52,465-472, and contact sensitizers J. Immunol. 166,3837-3845 B/I B story Adv. Cancer Res. 66,255-292[Medline] B and rel proteins in innate immunity Adv. Immunol. 58,1-27[Medline] B activation in hemopoietic progenitor cells J. Immunol. 160,1224-1232This article has been cited by other articles:
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  W. K. Nevala, C. M. Vachon, A. A. Leontovich, C. G. Scott, M. A. Thompson, S. N. Markovic, and for the Melanoma Study Group of the Mayo Clinic Ca Evidence of Systemic Th2-Driven Chronic Inflammation in Patients with Metastatic Melanoma Clin. Cancer Res., March 15, 2009; 15(6): 1931 - 1939. [Abstract] [Full Text] [PDF]
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