| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Immunology |
Charité,3 Department of Internal Medicine, Biomedizinisches Forschungszentrum, Campus Virchow, Humboldt University of Berlin, 13353 Berlin, Germany
IDEHU—Humoral Immunity Studies Institute-National Council of Scientific and Technological Research (CONICET),4 University of Buenos Aires, 1113 Buenos Aires, Argentina
Department of Microbiology,5 Parasitology, and Immunology, School of Medicine, University of Buenos Aires, Argentina
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
and major histocompatibility complex class II [MHC II] antigens), and presence of intracellular cytokines (interleukins 12 and 10) were determined by flow cytometry. The morphology of DC in the pregnant uterus was evaluated by immunohistochemistry. In uterus, the relative number of CD11c+ cells increased from GD 5.5, reaching a plateau on GD 9.5 until GD 17.5, while a transient peak of systemic CD11c+ cells was found on GD 8.5 and 10.5. The vast majority of uterine DCs were CD8- and thus, belonged to the myeloid lineage. Interestingly, a significant peak of lymphoid DC was present on GD 1.5 and 5.5. Further, significantly more intracellular interleukin 10 than interleukin 12 was present in CD11c+ cells. Interestingly, mature DCs (MHC II+) were diminished from GD 5.5 to 8.5. Characterization of CD11c+ cell kinetics in uterus and blood reveals variation of phenotype during pregnancy, pointing toward an eminent immunoregulatory role of DCs throughout gestation at the feto-maternal interface.
cytokines, decidua, immunology, implantation, pregnancy
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
In mice, DCs can be classified on the basis of their CD8 expression, whereby CD8-- and CD8+-DCs have been considered as myeloid and lymphoid, respectively [6]. However, it has been reported that both subpopulations can be generated from unique lymphoid committed precursor populations [7]. The CD8+ lymphoid DC subset is considered to prime naive CD4+ T cells to synthesize Th1 cytokines, such as interleukin (IL)-12, whereas the CD8- myeloid DC subset is thought to prime naive CD4+ T cells to produce Th2 cytokines, e.g., IL-10 [5, 8].
In pregnancy maintenance, several mechanisms are involved in the maternal tolerance against the fetus. Half a century ago, Medawar considered three mechanisms, which are still valid to date: 1) anatomic separation of fetus and mother, 2) antigenic immaturity of the fetus, and 3) immunologic tolerance of the mother [9]. The maternal balance between active immunity and tolerance at the feto-maternal interface, which is the site of contact between mother and child, is of crucial importance. Studies from Heikkinen et al. suggest that immunologic tolerance of the mother might be explained by the immunoinhibitory function of decidual macrophages at the feto-maternal interface [10].
However, because DCs constitute a complex system of cells uniquely able to commit between tolerogenic and immunogenic responses, they represent one major aspect to understand maternal tolerance. In fact, there is evidence that DCs could play a protective and immunoregulatory role at the feto-maternal interface. Kämmerer et al. described the presence of immunostimulatory mature DCs in decidual tissue during the early phase of human pregnancy. DCs seem to participate in the induction of decidual tolerance of the conceptus [11]. More recently, two pregnancy-protective molecules associated with DCs, CD200 and indoleamine 2,3-dioxygenase, have been suggested to play an immunoregulatory role at the maternal interface [12, 13].
Taking into account all this evidence, we hypothesized that DCs possess a regulatory function at the feto-maternal interface. Moreover, these cells might provide the signals deciding between allograft rejection and tolerance during gestation. Therefore, the aims of this study were
+ for lymphoid, and CD8- for myeloid, respectively. Furthermore, to evaluate the presence of major histocompatibility complex (MHC) II as a marker for DC maturity, and |
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Mice were purchased from Charles River (Sulzfeld, Germany) and maintained in an animal facility with a 12L:12D cycle. Animal care and experimental procedures were followed according to institutional guidelines and conformed to requirements of the state authority for animal research conduct (LaGetSi, Berlin). After overnight cohabitation of CBA/J females with DBA/2J males, females with vaginal plugs, which equals Day 0.5, were separated from the males and assigned to different groups and killed on Gestation Days (GDs) 1.5, 3.5, 5.5, 6.5, 7.5, 8.5, 10.5, 13.5, 15.5, or 17.5 (n = 7 in each GD). Also, we analyzed nonpregnant mice (n = 5). Random selection of these nonpregnant mice allowed us not to distinguish between stages of the estrous cycle of these mice, and thus, the data shown represent those for a pool of cells from animals either in estrus or not. Uteri were removed and divided into pieces, one section was frozen for immunochemistry, and resident uterine cells were isolated as described below.
Blood Cells
Blood samples were obtained by retro-orbital puncture shortly before the mice were killed. Blood was collected in heparinized tubes. After treatment with ammonium chloride lysis buffer for 10 min to deplete erythrocytes, the cells were washed twice with sterile phosphate-buffered saline (PBS).
Uterus Cell Isolation
After the mice were killed, the abdomen was carefully opened and access to the uterus was gained by pushing intestinal tissue to the side. The uterus was then removed by surgical cuts at the cervix and the ovaries. Then the uteri were fixed to a clamp at the cervix, which gave enough stability and allowed carefully cutting along the uterine horns. Then the embryos and placentas were cautiously peeled from the decidua. The remaining parts of the uterus, consisting of deciduas and myometrium, were washed in PBS to remove possible blood contamination. Then uterus-resident cells were isolated by a method described by Kämmerer et al. [11], with some modifications. In brief, the uterus was again washed with sterile PBS, carefully cut into small pieces, collected in tubes containing Hanks balanced salt solution (HBSS), and digested for 20 min at 37°C under slight agitation in HBSS with 200 U/ml hyaluronidase, 1 mg/ml collagenase type IV, 0.2 mg/ml DNase I, and 1 mg/ml bovine serum albumin/fraction V. All reagents were purchased from Sigma (Taufkirchen, Germany). Thereafter, the isolated cells were collected in a fresh tube through a 100-µm net (BD Biosciences, San Jose, CA) and washed with RPMI (developed at the Roswell Park Memorial Institute [Buffalo, New York], hence the acronym is RPMI; purchased at Gibco, Invitrogen, Eggenstein, Germany)-10% fetal bovine serum (FBS). The procedure was repeated twice, with HBSS medium containing no cocktail of enzymes. Cell populations were obtained by density-gradient centrifugation in a 1.088 g/ml Lympholyte-M solution (Cedarlane Labs, Hornby, ON, Canada) upon centrifugation at 2400 rpm for 20 min at room temperature (RT). The low-density fraction at the interface between Lympholyte-M and medium was collected and washed several times. We then counted the total number of uterine cells and observed some variations between the different days of gestation; however, significant differences of isolated cells per milliliter of cell suspension between the various days of gestation could not be detected.
Flow Cytometric Analysis
For flow cytometry, the uterine and blood cells were incubated for 3 h with Brefeldin A (106 cells/ml medium with 1 µl/ml of Golgi Plug; BD Pharmingen, Heidelberg, Germany) in RPMI with FBS in a humidified incubator at 37°C with 5% CO2. Brefeldin A is a commonly used agent that blocks cytokine secretion through inhibition of the Golgi apparatus. Flow cytometry was performed as follows: for immunostaining, uterus cells were washed twice with ice-cold PBS supplemented with 1% bovine serum albumin (Sigma) and 0.1% Natrium Azid (Sigma). Two percent of normal mouse serum was added to avoid nonspecific binding by Fc receptors. Cells were then incubated 30 min at 4°C with previously optimized amounts of one or more of the following conjugated murine monoclonal antibodies (Mabs): anti-CD8a fluorescein isothyocianate, anti-Ie-K (MHC II)-PE, and the biotinylated Mab anti-CD11c. All Mabs were purchased from BD Biosciences. Streptavidin-PerCP was used as a second-step reagent. Cells were washed, fixed using Fix solution (Becton Dickinson, Erembodegem, Belgium), and incubated for 20 min at RT in the dark. Subsequently, the cells were washed and permeabilized, using fluorescence activated cell sorting (FACS) permeabilizing solution (Becton Dickinson), followed by incubation with intracellular antibody PE-labeled IL-12 and IL-10 for 30 min at 4°C in the dark. As controls, cells were stained with the corresponding isotype-matched Mab. The cells were then washed and analyzed. The acquisition was performed using a FACS Calibur (Becton Dickinson). A minimum of 30 000 events per analysis were examined. Instrument compensation was set in each experiment by single-color stained samples. Data were analyzed by Cell Quest software. Flow cytometry results were expressed as percentage of cells positive for the surface marker and for intracellular cytokines.
Immunohistochemical Staining for CD11c+
Eight-micron cryosections were incubated with avidin- and biotin-blocking solution (Vector Laboratories, Burlingame, CA), followed by another blockade step using protein blocking agent (Immunotech, Beckman Coulter, Krefeld, Germany). The biotinylated hamster-anti-mouse CD11c Mab (BD Biosciences) was diluted 1:100 in Tris-buffered saline (TBS) containing 1% FBS and applied for 1 h. As the amplification and developing system, we used avidin-biotin-alkaline phosphate complex (Vector Laboratories) 1:100 in TBS for 30 min. After washing, a routine staining procedure for alkaline phosphate was used and sections were counterstained with Mayer hematoxylin. Thereafter, sections were covered with Kaiser glycerol gelatin. Slides were examined using a Zeiss Axioscope light microscope (Carl Zeiss Inc., Oberkochen, Germany). Photo documentation was performed using a digital image analysis system (Zeiss KS400).
Statistical Analysis
Statistical significance was determined using the nonparametric Mann-Whitney U-test for comparison of two samples. The observations over time were tested with the Kruskal-Wallis test. The results are presented as mean and SD. Differences at P < 0.05 are considered statistically significant.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Because murine DC subsets can be identified by their expression of CD11c on the cell surface [14], we first focused on the expression of this distinct DC cell surface molecule on uterus and blood cells. The percentages of CD11c+ cells at the uterus and in the blood of nonpregnant mice were not different from those of pregnant mice on GD 1.5 (Fig. 1). CD11c expression in uterine cells significantly increased on completion of implantation (GD 5.5), reaching a plateau of relative CD11c+ cell numbers of 10% among the resident uterine cells. In general, the percentage of cells expressing CD11c was lower in blood than in the uterus during pregnancy (see Fig. 1 and Table 1). In blood, we found an increase of CD11c expression on GD 8.5 and 10.5 compared with the other evaluated GDs (see Table 1).
|
|
Lymphoid/Myeloid Lineage of CD11c+ Cells in Uterus and Blood
Next, we determined, again by flow cytometry, the expression of CD8 in uterine and blood CD11c+ cells. In the uterus, the vast majority of CD11c+ cells are CD8-; thus, they pertain to the myeloid lineage. Nevertheless, a significant and transient shift toward a lymphoid phenotype of uterine DCs could be detected on GDs 1.5 and 5.5 (Fig. 2).
|
Also in the blood, the percentage of myeloid CD11c+ cells, as identified by CD8 negativity, was predominant. The highest levels of lymphoid CD11c+ were observed on GDs 10.5 and 13.5 (see Table 1), which were significantly different compared with the other investigated GDs.
Expression of MHC Class II Molecules by CD11c+ Cells
Further, we investigated whether CD11c+ cells coexpressed MHC class II molecules during gestation, which may then be considered as mature DCs. Indeed, we found that CD11c+ from blood and uterine cells spontaneously express MHC class II throughout pregnancy (see Fig. 3 and Table 1). In the uterus, less than 30% of resident CD11c+ cells expressed MHC class II molecules; thus, the majority of uterine DCs are immature. Surprisingly, from GD 5.5 to GD 8.5, the expression of MHC class II molecules on the surface of uterine CD11c+ cells was significantly reduced.
|
A high percentage of MHC class II expressing CD11c+ cells in blood could be detected late in gestation (GDs 15.5 and 17.5).
Cytokine Expression in CD11c+ Cells
Orientation of immune responses by DCs is directly related to their profile of cytokines. Therefore, we studied the expression of IL-12 as a Th1 marker/inducer and IL-10 as a Th2 marker in uterine and blood CD11c+ cells during pregnancy (Fig. 4 and Table 2).
|
|
The relative number of IL-12-producing CD11c+ cells in uterus was constantly lower than in the blood. In the uterus, the intracellular presence of IL-10 was significantly higher than the expression of IL-12 in CD11c+ cells, with the exception of GD 5.5.
In the blood, IL-10-producing DCs predominate mildly over IL-12-producing DCs. However, on GDs 5.5, 10.5, and 13.5, the domination of IL-10 over IL-12 increased significantly.
Tissue Distribution of CD11c Expression During Pregnancy
To further characterize CD11c+ DC cells, we performed immunohistochemical staining on frozen uterus tissue sections (Fig. 5). CD11c+-expressing cells were of ovoid and macrophage shape. These positive cells could be detected in uterine as well as in placental tissue. Interestingly, they did not differ morphologically from each other.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-) over lymphoid (CD8+) phenotype of uterine DCs and why do DCs express higher levels of Th2 (IL-10) than Th1 (IL-12) cytokines? In the present study, we observed that the relative number of CD11c+ uterine cells is not constant during pregnancy. Indeed, we found an increase of CD11c+ uterus cells from GD 5.5, with the highest point of expression on GD 8.5, reaching a plateau on GD 9.5 until GD 17.5. The increment of CD11c+ uterine cells occurs simultaneously with the decisive phase of gestation, when implantation takes place. In fact, Gorczynski et al. [12] recently described that CD200, a further molecule expressed on DCs, is associated with the maintenance of pregnancy in allogeneic mouse models, such as the CBA/J x BALB/c model [15] and the CBA/J x DBA/2J model, which has been referred to as abortion prone because an increased abortion rate can be induced by application of lipopolysaccharide, Th1 cytokines, or stress exposure [12, 15]. Taking this into account, we suggest that DCs are involved in the immunological events that accompany the maintenance of mammalian pregnancy at the time of implantation, which points toward understanding discussion aspect 1.
The different murine DC subtypes share a common capacity to present antigens to T cells and promote cell-cycle progression. However, they differ in some aspects of DC-T-cell signaling that determine the type of immune response. Thus, CD8+ DCs have the ability to induce a Th1-biased cytokine response, whereas CD8- DCs are prone to induce a Th2-biased response [16–18].
Cytokines are known to regulate the rejection or maintenance of pregnancy. Th1 cytokines (IL-2, tumor necrosis factor , IL-12, interferon 
) have been shown to boost the abortion rate, while Th2 cytokines (IL-3, IL-4, IL-10) appear to be pregnancy-protective [19–21]. The balance between Th1 and Th2 is crucial for successful pregnancy, whereby the temporal positioning of Th1-type and Th2-type cytokines and their relative concentrations appear to be quite critical. It is generally accepted that some Th1-type cytokines are involved in mediating the communication between embryonic cells and maternal uterine cells, as part of the physiologic response to implantation [22, 23]. Thus, it is crucial to know whether and when DCs produce IL-10 and/or IL-12 cytokines in the specific environment of the feto-maternal interface. Indeed, the production of IL-12 by DCs is required to induce T-cell priming and might represent a switch to immunogenicity, whereas IL-10 production by these cells is supposed to induce a tolerogenic (or at least regulatory) and thus pregnancy-protective response [16–18].
In this study, we report that the majority of uterine DCs were myeloid, as identified by their negativity for CD8 supporting the pregnancy-protective role of DCs. Furthermore, we determined that intracellular presence of IL-10 by DC was significantly higher than IL-12 during most of the gestation. With respect to discussion point 2, this bias toward IL-10 production at the uterine level seems to be the key to induction of tolerance because this cytokine acts as an inhibitor of the immune response in different ways: by suppressing T cells itself [24]; by inducing differentiation to regulatory T cells [25, 26]; or by inhibiting DC terminal differentiation [27, 28], skewing them toward a tolerogenic phenotype [29].
However, another interesting finding of this study was the transient increase in the percentage of CD8+ DC uterine cells in early gestation, namely on GD 5.5, which was accompanied by a transitory decrease in the relative number of IL-10-producing DCs. The remarkable plasticity in the ability to induce Th1 or Th2 cytokines, temporal regulation, and subtle discrimination of antigen stimuli by DCs has been recognized [30]. Therefore, we suggest that fetal antigens could be stimulating CD8+ DC uterine cells to produce Th1 cytokines during the peri-implantation period of pregnancy. Thus, DCs seem to contribute to the development of an adequate environment during the implantation period. Afterward, a Th2-biased cytokine pattern is predominant, which mediates the communication between embryonic cells and maternal uterine cells and the establishment of pregnancy [19–23]. We cannot conclude that CD8+ DCs are responsible for the production of Th1-cytokines during peri-implantation because recently published data demonstrated disparate roles of DC subsets in various experimental models [31, 32]; nevertheless, this observation might provide a clue regarding discussion point 2.
Furthermore, we observed that some part of uterine DCs constitutively express MHC class II, indicating that these resident DCs possess some degree of maturation. Recently, Colledge and coworkers reported that constitutive peptide-MHC class II complex generation occurs in DCs in immunologically quiescent situations and that antigen presentation by nonactivated DCs might play a role in the induction of tolerance [33]. Worthy of note is the finding of a downregulation of MHC class II expression on the surface of uterine DCs from GD 5.5 until GD 8.5. A recent review by Lutz and Schuler [4] about the state of DC maturation and the ability to induce tolerance or immunity gives some hints to understand this phenomenon: according to their model, DCs expressing low quantities of MHC class II are supposed to induce T-cell anergy in the absence of antigen stimulation. Therefore, the observation of DCs expressing low levels of MHC class II during this period of gestation might be related to fetal acceptance, which might give an explication to question 3.
Comparing the relative numbers of CD11c+ cells in blood and locally, we observed lower cell numbers for CD11c+ cells in blood, suggesting a focal point of function for DCs in the local environment. This agrees with published data where DCs are considered to be responsible for tolerance of antigens in mucosal surfaces [34]. Moreover, the phenotype of blood DCs revealed a higher grade of maturation compared with uterine DCs as identified by their expression of MHC II molecules and the total amount of cytokine production. This disparity of the maturation status between blood and uterine DCs suggests different roles in the maintenance of pregnancy between blood and locally in the uterus and might provide an explanation of question 4.
In conclusion, we have characterized the phenotype and function of CD11c+ cells in the uterus and in blood during pregnancy. Features described here, as the varying number of resident DC subsets, changes in the expression pattern of IL-10 and IL-12 cytokines and MHC II molecules by DCs, demonstrate that DCs represent a cell population with unique properties required for the maternal tolerance of the fetus. Therefore, this study provides a basis for further research and suggests that examining the function of DCs and DC subsets may be relevant to further understanding of the immunology at the feto-maternal interface.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Correspondence: Petra Clara Arck, Biomedizinisches Forschungszentrum, Raum 2.0549, Augustenburger Platz 1, 13353 Berlin, Germany. FAX: +49 30 450 553962; petra.arck{at}charite.de
Received: 28 August 2003.
First decision: 3 October 2003.
Accepted: 3 December 2003.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
+ and CD8- subclasses of dendritic cells direct the development of distinct T helper cells in vivo. J Exp Med 1999 189:587-592- and CD8+ dendritic cells generated from CD4low lymphoid-committed precursors. Blood 2000 96:2511-2519 and IL-1 block stress-triggered murine abortion. Am J Reprod Immunol 1997 37:262-268This article has been cited by other articles:
|
|
 |
|
  J. Behrends, C. M. Karsten, S. Wilke, A. Robke, and A. Kruse Identification of ITGA4/ITGB7 and ITGAE/ITGB7 Expressing Subsets of Decidual Dendritic-Like Cells Within Distinct Microdomains of the Pregnant Mouse Uterus Biol Reprod, October 1, 2008; 79(4): 624 - 632. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W.-H. Zhou, L. Dong, M.-R. Du, X.-Y. Zhu, and D.-J. Li Cyclosporin A improves murine pregnancy outcome in abortion-prone matings: involvement of CD80/86 and CD28/CTLA-4 Reproduction, March 1, 2008; 135(3): 385 - 395. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. M Blois, U. Kammerer, C. A. Soto, M. C Tometten, V. Shaikly, G. Barrientos, R. Jurd, D. Rukavina, A. W Thomson, B. F Klapp, et al. Dendritic Cells: Key to Fetal Tolerance? Biol Reprod, October 1, 2007; 77(4): 590 - 598. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A.-H. Zarnani, S.-M. Moazzeni, F. Shokri, M. Salehnia, and M. Jeddi-Tehrani Kinetics of murine decidual dendritic cells Reproduction, January 1, 2007; 133(1): 275 - 283. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Tometten, S. Blois, A. Kuhlmei, A. Stretz, B. F. Klapp, and P. C. Arck Nerve Growth Factor Translates Stress Response and Subsequent Murine Abortion via Adhesion Molecule-Dependent Pathways Biol Reprod, April 1, 2006; 74(4): 674 - 683. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Lima, V. M. O. Souza, E. L. Faquim-Mauro, M. S. Hoshida, E. Bevilacqua, M. S. Macedo, W. Tavares-de-Lima, and B. B. Vargaftig Modulation of the Induction of Lung and Airway Allergy in the Offspring of IFN-{gamma}-Treated Mother Mice J. Immunol., September 15, 2005; 175(6): 3554 - 3559. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Blois, M. Tometten, J. Kandil, E. Hagen, B. F. Klapp, R. A. Margni, and P. C. Arck Intercellular Adhesion Molecule-1/LFA-1 Cross Talk Is a Proximate Mediator Capable of Disrupting Immune Integration and Tolerance Mechanism at the Feto-Maternal Interface in Murine Pregnancies J. Immunol., February 15, 2005; 174(4): 1820 - 1829. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
0.05; **, with P 
