Published online before print July 15, 2003
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Departments of
* Obstetrics and Gynecology and
Pediatrics, Toyama Medical and Pharmaceutical University, Japan
1 Correspondence: Department of Obstetrics and Gynecology, Toyama Medical and Pharmaceutical University, 2630 Sugitani Toyama-shi, Toyama 930-0194, Japan. E-mail: s30saito{at}ms.toyama-mpu.ac.jp
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: dendritic cell • IL-12 • myeloid DC • pregancy • Th2
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Dendritic cells (DCs) possess some unique properties that enable them to sensitize naïve T cells in vitro and in vivo [9 10 11 ]. Conversely, interleukin-10 (IL-10)-treated, immature DCs induce a state of alloantigen-specific anergy in CD4+ T cells [12 13 14 ]. Thus, DCs have the capability to perform two roles: antigen presentation and the induction of peripheral tolerance.
Naïve T cells differentiate into discrete subsets of cytokine-secreting cells, such as those represented by the T helper cell type 1 (Th1) and Th2 phenotypes [15 ]. Recent findings demonstrated that DCs derived from the myeloid lineage could promote Th1 responses, and DCs derived from the lymphoid lineage could promote Th2 responses [16 17 18 ]. Whelan et al. [19 ] reported that the switch to Th1 or Th2 responses is not affected by differential regulation through costimulatory molecules such as CD80 or CD86 and that a Th1 response is achieved in the presence of IL-12. In the human endometrium and decidua, the Th1/Th2 balance quickly changes from a Th1 dominant state to a Th2 dominant state after pregnancy [20 , 21 ]. An excess of type 1 activity in the implantation site is emerging as a key feature of pregnancy disorders suggested to have an immunologic etiology, including spontaneous abortion and pre-eclampsia [22 23 24 ]. Therefore, DCs could be suitable candidates for the mediators that balance maternal immunostimulation, tolerance, and the Th1/Th2 balance. In this study, we examined the expression of the surface markers on decidual DCs and IL-12 production by decidual DCs using flow cytometry. The present findings showed that decidual DCs express mature, costimulatory markers such as CD80 and CD86, suggesting that decidual DCs play a role in antigen presentation. It is interesting that we show that the majority of decidual DCs were myeloid DCs, which had less ability to produce IL-12 and induced the differentiation of Th2 responses. Thus, decidual DCs have unique, immunologic characteristics that differ from the peripheral blood DCs and might regulate the Th1/Th2 balance in the uterus.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PBMC and decidual mononuclear cell preparations
PBMC were isolated by the standard Ficoll-Hypaque method. Decidual samples obtained from induced abortion cases were separated carefully from villi under a stereomicroscope. The decidual mononuclear cells (leukocytes) were purified by the Ficoll-Hypaque method after homogenization and filtration through a 32-µm nylon mesh, as previously reported [5
, 6
]. PBMC and decidual mononuclear cells were obtained from the same induced abortion cases. Informed consent was obtained from all subjects.
Reagents
The following materials were obtained from Becton Dickinson (San Jose, CA): fluorescein isothiocyanate (FITC)-conjugated lineage cocktail mouse monoclonal antibodies (mAb) to CD3, CD14, CD16, CD19, CD20, and CD56; phycoerythrin (PE)-conjugated mouse mAb to CD123 [anti-IL-3 receptor (R)
], CD11c. PE-conjugated CD83 was obtained from Immunotech (Marseille, France); peridinin chlorophyll protein (Per CP)-conjugated mouse mAb to HLA-DR; allo-phycocyanin (APC)-conjugated mouse mAb to CD11c and HLA-DR; and biotin-conjugated mouse mAb to CD80, CD86, and human IL-12 p70.
Flow cytometric analysis
The PBMC and decidual mononuclear cells from 14 subjects were suspended in phosphate-buffered saline (PBS) containing 1% fetal calf serum and 0.02% sodium azide. Cells were incubated for 25 min in the dark at room temperature with FITC-conjugated lineage cocktail mAb, Per CP-conjugated anti-HLA-DR mAb, PE-conjugated mAb to CD123, and APC-conjugated mAb to CD11c. The cells from the other subjects were incubated for 25 min in the dark at room temperature with FITC-conjugated lineage cocktail mAb, Per CP conjugated with anti-HLA-DR mAb, PE-conjugated mAb to CD83, and biotin-conjugated mAb to CD80 or CD86. After washing, the cells were incubated with RED670-conjugated streptavidin (Life Technologies, Gaithersberg, MD) for an additional 30 min and analyzed on a FACSCalibur cytofluorimeter (Becton Dickinson).
Staining for intracellular cytokines and surface antigens for flow cytometry
Intracellular cytokines were stained according to the method of Picker et al. [25
] with some modifications. Briefly, the peripheral blood and decidual leukocytes from seven subjects were stimulated with phorbol myristate acetate (PMA; 20 ng/ml) and ionomycin (250 ng/ml) in the presence of brefeldin A (10 µg/ml) for 4 h. These mononuclear cells were stained with FITC-conjugated lineage cocktail mAb, APC-conjugated anti-HLA-DR mAb, and PE-conjugated CD11c or CD123 mAb. Cells were washed and fixed in 4% formaldehyde/PBS at room temperature for 5 min and then again washed and treated with permeabilizing solution (Becton Dickinson) at room temperature for 10 min. These fixed and permeabilized mononuclear cells were stained with biotin-conjugated anti-human IL-12 p70 mAb, washed, and then incubated with RED670-conjugated streptavidin for 30 min. Cells were analyzed on a FACSCalibur cytofluorimeter using CellQuest software (Becton Dickinson).
Purification of DCs and CD4+CD45RO- naïve T cells
DCs were isolated from PBMC and decidual mononuclear cells by magnetic cell sorting using a CD1c (blood DC antigen-1) DC isolation kit (Miltenyi Biotec, Bergish, Glandbach, Germany). To obtain CD45RO- cells, PBMC isolated from healthy, nonpregnant women were incubated with anti-CD45RO mAb-conjugated MicroBeads (Miltenyi Biotec), and CD45RO-positive cells were removed by magnetic cell sorting, according to the manufacturer’s instruction. Subsequently, CD45RO- cells were incubated with anti-CD4-conjugated MicroBeads (Miltenyi Biotec). Magnetic cell sorting purified CD4+CD45RO- naïve T cells. The resulting populations were >95% lin-CD11c+ DCs and >95% CD4+CD45RO- naïve T cells, respectively.
Stimulation of DCs
DCs (10x103/well) were stimulated in 96-well, U-bottomed, tissue-culture plates by Staphylococcus aureaus Cowan 1 strain (SAC; 0.01% vol/vol; Pansorbin, Calbiochem, San Diego, CA), lipopolysaccharide (LPS; 1 µg/ml; from Escherichia coli serotype 055:B5; Sigma Chemical Co., St. Louis, MO), and CD40L-transfected L cells (1.25x106/well; provided by Dr. Yung-Jun Liu, DNAX Research Institute, Palo Alto, CA) in 200 µl RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (Hyclone, Logan, UT), 1% penicillin-streptomycin (Gibco-BRL, Grand Island, NY), and 1.0 µg/ml fungizone (Gibco-BRL). All modes of stimulation were performed in the absence or presence of interferon-
(IFN-; 500 U/ml; R&D Systems, Minneapolis, MN). Supernatants were collected after 24 h, and IL-12 p70 levels in the supernatants were analyzed by the enzyme-linked immunosorbent assay (ELISA) method.
Mixed leukocyte reaction (MLR) and intracellular cytokines analysis
Primary MLRs were conducted in 96-well, U-bottomed, tissue-culture plates by adding mitomycin C-treated DCs in 200 µl complete culture medium in the presence or absence of 1 ng/ml recombinant IL-12 (rIL-12; R&D Systems) at a 1:100 stimulator (DCs)/responder (T cells) ratio. After 4 days of culture, 100 µl culture supernatant was replaced with fresh medium containing 100 U/ml rIL-2 (R&D Systems). On days 6 and 8, cultures were split and expanded in the presence of 50 U/ml rIL-2. After 7–10 days of rIL-2 expansion, T cells were washed and stimulated with PMA (20 ng/ml) and ionomycin (250 ng/ml) in the presence of brefeldin A (10 µg/ml) for 4 h. Cells were washed, fixed, and permeabilized. Intracellular cytokines were stained with FITC-conjugated IFN- and PE-conjugated IL-4 (Becton Dickinson) and analyzed on a FACSCalibur cytofluorimeter using CellQuest software (Becton Dickinson).
Cytokines quantitation
ELISA kits (R&D Systems) were used to quantify human IL-12 p70 in supernatants.
Statistical analysis
Paired t-test, Student’s t-test, and Mann-Whitney U-test analyzed the data. A P< 0.05 was considered significant.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Figure 1. Expression of lineage antigens (CD3, CD14, CD16, CD19, CD20, and CD56), HLA-DR, and CD45 on decidual leukocytes. Decidual leukocytes were analyzed by flow cytometry with FITC-labeled lineage cocktail mAb and PE-labeled mAb to HLA-DR in the presence (C) or absence (B) of FITC-labeled mAb to CD45. (A) A gate was set on leukocytes using characteristic forward- and side-scatter parameters (R1; FSC and SSC, respectively). The majority of lin-HLA-DRbright cells (boxR2) shifted to boxR3 in the presence of FITC-labeled mAb to CD45, suggesting that the majority of decidual lin-HLA-DRbright cells expressed the common leukocyte antigen CD45.
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Table 1. Percentages of Myeloid DCs and Lymphoid DCs in Peripheral Blood and in Decidua during Early Pregnancy
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Figure 2. Expression of CD80, CD83, and CD86 on peripheral blood DCs and decidual leukocytes. Cells were stained with FITC-conjugated lineage cocktail mAb, Per CP-conjugated mAb to HLA-DR, PE-conjugated mAb to CD83, and biotin-conjugated mAb to CD80 or CD86. After washing, the cells were incubated with RED670-conjugated streptavidine. The expressions of CD80, CD83, or CD86 on lin-HLA-DRbright (R2) are displayed.
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Figure 4. Expression of CD11c and CD123 on peripheral blood DCs and decidual DCs. Cells were stained with FITC-conjugated lineage cocktail mAb, Per CP-conjugated mAb to HLA-DR, APC-conjugated mAb to CD11c, and PE-conjugated mAb to CD123. A gate was set on the leukocytes (R1) as described in Figure 1
. The expression of CD11c and CD123 on lin-HLA-DRbright DCs (R2) is displayed in the right panel.
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MFI), MFI of total decidual DCs was significantly lower than that of peripheral blood DCs (15.72±7.53 vs. 32.79±6.90; P=0.016). MFI of the decidual myeloid DCs (18.38±7.62) was significantly lower than that of peripheral blood myeloid DCs (49.27±16.46; P=0.009; Fig. 5B
). MFI of the lymphoid DCs was not determined, as those events were very small. Next, we checked the production of IL-12 p70 by activated myeloid DCs (Fig. 6
). IL-12 production by decidual DCs stimulated with CD40L, LPS, SAC, or IFN- plus SAC was significantly lower than that by peripheral myeloid DCs.
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Figure 5. Intracytoplasmic IL-12 in peripheral blood DCs and decidual DCs. (A) A gate was set on leukocytes, and the presence of intracytoplasmic IL-12 in lin-HLA-DRbright cells was examined. IL-12 production in CD123+ DCs and CD11c+ DCs is displayed. (B) Data are presented as histgrams. Broken line, Negative-control Ab staining; solid line, staining of intracytoplasmic IL-12.
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Table 2. The Percentage of IL-12 Producing Myeloid DCs and Lymphoid DCs in the Decidua and Peripheral Blood
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Figure 6. Production of IL-12 by peripheral blood DCs (PBL) and decidual DCs (DEC). DCs were stimulated with CD40L, LPS, SAC, or SAC plus IFN-. Supernatants were collected after 24 h, and the levels of IL-12 were assayed. Results are presented as means ± SEM of four independent experiments. *, Significantly different from decidual myeloid DCs by Mann-Whitney U-test; P< 0.05.
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) on T cells by flow cytometry. Naïve T cells primed with decidual DCs led to a higher percentage of IL-4-producing cells (Th2 cells) in comparison with that with peripheral myeloid DCs. The Th1/Th2 ratio primed with decidual myeloid DCs was 3.38 ± 2.04. That ratio was significantly lower than that primed with peripheral myeloid DCs (Table 3
). Thus, decidual myeloid DCs drive T cell polarization toward a Th2 phenotype. Conversely, the percentage of Th2 cells, which were primed with decidual myeloid DCs cultured with rIL-12, decreased to 4.37 ± 1.88%, and this level was similar to that which primed peripheral blood myeloid DCs (Table 4
; Fig. 7
). Treatment with rIL-12 significantly induced Th1 cells primed with decidual myeloid DC from 24.24 ± 7.57% to 47.74 ± 17.07%. As a result, the Th1/Th2 ratio primed with decidual myeloid DC with rIL-12 increased to 14.22 ± 11.65 (Table 4)
. Thus, exogenous rIL-12 administration with decidual myeloid DCs abolished the development of Th2 cells. |
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Table 3. Development of Th0, Th1, and Th2 Cells Promoted by Decidual Myeloid DCs and Peripheral Blood Myeloid DCs
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Table 4. Development of Th0, Th1, and Th2 Cells Promoted by Decidual Myeloid DCs and Peripheral Blood Myeloid DCs by Adding Exogenous IL-12
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Figure 7. Expression of IL-4 and IFN- in Th cells promoted by decidual DCs or peripheral blood DCs. Decidual myeloid DCs and peripheral myeloid DCs were cultured with naïve allogenic CD4+CD45RO- T cells in the absence of rIL-12 (upper panels) or in the presence of rIL-12 (lower panels). We analyzed the pattern of intracellular cytokines (IL-4 and IFN-) in T cells by flow cytometry.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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At the moment of priming, naïve Th cells can receive an initial Th1 or Th2 polarizing signal. Recent findings suggested that a component of this early polarizing signal can be carried by DCs that originate from the site of pathogen entry and are functionally modified by local conditions [16
, 17
, 19
, 28
]. Two distinct lineages of DC have been described in humans [17
, 18
, 28
, 29
]. Myeloid DCs express myeloid antigens CD11c, CD13, and CD33. In human peripheral blood, myeloid DCs were identified as lin-HLA-DRbright CD11c+. Myeloid DCs produce high levels of IL-12 when stimulated with tumor necrosis factor (TNF-) or CD40L and drive T cell differentiation into Th1 [17
, 28
, 30
]. Lymphoid DCs lack myeloid markers and have IL-3R (CD123). Lymphoid DCs can induce T cell differentiation into Th2 cells [17
18
19
], and lymphoid DCs were suggested to perform mainly a tolerogenic function [29
] as a result of their ability to induce apoptosis in responsive Th cells [31
].
Although the physiologic protection from fetal rejection was suggested to be a result of a Th2-type response at the fetomaternal interface [20 , 32 , 33 ], the present findings demonstrated that myeloid DCs were predominant in the decidua. Until recently, myeloid DCs were regarded as Th1-driving antigen-presenting cells. However, several in vitro and in vivo studies have shown that myeloid DCs are good inducers of Th1 and Th2 cells [16 , 34 , 35 ]. Recent findings also have demonstrated that the levels of IL-12 produced by myeloid DCs can provide Th1 or Th2 polarity [16 , 34 , 36 ]. The present findings clearly demonstrated that the percentages of IL-12 producing DCs, myeloid DCs, and lymphoid DCs were significantly lower in the decidua than those in the peripheral blood. In addition, IL-12 secretion by decidual myeloid DCs stimulated with LPS, SAC, or CD40L was significantly lower than that by peripheral blood myeloid DCs. Furthermore, when rIL-12 was added in culture media, the percentage of Th2 primed with decidual myeloid DCs decreased, and the ratio of Th1/Th2 increased. This decreased IL-12 production by decidual myeloid DCs could facilitate optimal Th2 response development in decidua.
It was reported that the ability of human myeloid DCs to promote Th1 or Th2 differentiation is critically dependent on the stimulator/responder ratio [37
]. We defined the stimulator/responder ratio as 1:100, as the percentage of DCs in peripheral blood and decidua was 
1%. In this condition, decidual myeloid DCs induced more Th2 cells compared with that by peripheral blood myeloid DCs. Ebner et al. [38
] reported that immature [CD83 (-), lysosome-associated membrane glycoprotein (-)] myeloid DCs stimulated with CD40L or bacteria always secreted more IL-12 than already mature [CD83 (+), lysosome-associated membrane glycoprotein (+)] myeloid DCs. In the present study, expression of CD83 on decidual DCs was similar to that on peripheral blood DCs (Fig. 3)
, suggesting that decreased IL-12 production by decidual myeloid DCs did not result from the maturation of DCs. Liu et al. [39
] reported that estrogen decreased TNF-, IFN-, and IL-12 production in mature DCs. High estrogen levels at the fetomaternal interface could have effects on decidual myeloid DCs to reduce IL-12 production. Prostaglandin E2 (PGE2) and IL-10 inhibit IL-12 production in immature DCs [40
, 41
]. IL-10 prevents DC development when present during the early stages; conversely, during the final stage of DC maturation, the presence of PGE2 results in type 2-polarized effector cells by modulating IL-12 production [16
]. As IL-10 and PGE2 are present in decidua, these molecules might inhibit IL-12 production by the decidual myeloid DCs, thereby shifting the Th1/Th2 balance to a Th2 dominant state at the fetomaternal interface.
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Figure 3. Comparison of CD80+, CD86+, and CD83+ decidual DCs and peripheral blood DCs. Dots linked with a line represent samples from the same individual.
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Decidual DCs express the costimulatory molecules CD80 and 86 and HLA-DR, and they have the ability to present antigen. However, there must be some mechanism to induce T cell tolerance. As one possible mechanism, via a Th2-type cytokine, IL-10-treated DCs might induce tolerance. At the fetomaternal interface, extravillous trophoblasts and Th2 cells produce IL-10 [33 , 44 ], and this IL-10 might induce alloantigen (fetal antigen)-specific anergy.
In conclusion, DCs were present in early pregnancy decidua. The majority of the DC population was myeloid DCs in the deciduas; however, IL-12-producing myeloid DCs were decreased in the decidua. Decidual DCs could regulate the Th1/Th2 balance to maintain a Th2 dominant state, leading to maintenance of pregnancy.
Received November 18, 2002; revised April 6, 2003; accepted June 4, 2003.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and IL-2Rß on T cells of human decidua at an early stage of pregnancy Immunology 75,710-712[Medline]+CD83+ dendritic cells: the levels of IL-12 are determined during the final dendritic cell maturation and are resistant to further modulation J. Immunol. 161,2804-2809This article has been cited by other articles:
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