1 Department of Physiology and Immunology, School of Medicine, University of Rijeka, B. Branchetta 20/1,2 Department of Obstetrics and Gynaecology, Clinical Hospital Centre, University of Rijeka, Cambierrieva 42, 51000 Rijeka, Croatia and 3 Department of Immunology and Cell Biology, Mario Negri Institute for Pharmacological Research, Via Eritrea 62, 20157 Milan, Italy
4 To whom correspondence should be addressed at: Department of Physiology and Immunology, Medical Faculty, University of Rijeka, B. Branchetta 20/1, HR-51000 Rijeka, Croatia. Email: gordana.laskarin{at}medri.hr
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Abstract |
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Key words: decidua/macrophages/mannose receptor/term pregnancy/tumor associated glycoprotein-72
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Introduction |
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Extensive remodeling events occur during embryo implantation and early pregnancy, comprising trophoblast invasion, infiltration of decidua by maternal immunocompetent NK cells, T lymphocytes and antigen presenting cells (macrophages and dendritic cells), differentiation of glandular epithelium and transformation of spiral arteries into uteroplacental vessels (Fazleabas and Strakova, 2002; Trundley and Moffett, 2004
). Antigens of paternal origin expressed on trophoblast cells come into close contact with maternal leukocytes present in the decidua. These interactions, instead of bringing about a detrimental immune response, cause tolerance supported by progesterone (Szekeres-Bartho and Wegmann, 1996
; Szekeres-Bartho et al., 2001
) and set of complex cytokine and chemokine production (Jerzak and Bischof, 2002
; Piccinni, 2002
; Miyazaki et al., 2003
). Recent data show that engaging of the CRD of the MR by specific antibody, as well as glycoproteins, could lead to triggering of an anti-inflammatory and tolerogenic program in dendritic cells (Chieppa et al., 2003
). The interaction of MR with glycoproteins at the maternalfetal interface could promote Th2 cytokine production, indispensable for maintaining pregnancy, following the narrow peri-implantation period (Chaouat et al., 1999; Chaouat et al., 2003
). Although tumor associated glycoprotein-72 (TAG-72) is the marker of various human malignancies (Katari et al., 1990
), it is produced throughout normal human female genital tract (Osteen et al., 1992
) in a progesterone dependent manner (Osteen et al., 1990
). With its high glycosylation level, TAG-72 could be possible natural ligand for the MR. Although the MR protein was originally isolated from human placenta (Lennartz et al. 1987), little is known about the distribution and function of this receptor at the maternalfetal interface, which is rich in progesterone (3 µg/g of decidual tissue) (Szekeres-Bartho, 1992
). The aim of our study was to analyze expression and distribution of the MR in the first trimester pregnancy decidua and term placenta, MR endocytic activity and possible binding properties of progesterone dependent TAG-72 to the CRD of the MR.
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Materials and methods |
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Immunohistology
Samples of the first trimester decidua and term placenta were snap frozen, cut into tissue sections and fixed in cold acetone (Kemika, Zagreb, Croatia). Sections of both tissues were stained by primary mouse immunoglobulin G (IgG) 2a anti-CD14 monoclonal antibody (anti-CD14 mAb) (School of Medicine, Rijeka, Croatia) and/or mouse anti-human anti-MR monoclonal antibody of PAM-1 clone (PAM-1 anti-MR mAb) (Mario Negri Institute for Farmacological Research, Milan, Italy, Chieppa-Wechii) using DAKO Envision Doublestain System (DAKO, Glostrup, Denmark). The manufacturer's instructions were followed for performing an indirect immunoperoxidase and alkaline phosphatase (AP)-staining methods. Briefly, sections pretreated with kit blocking solution were incubated (30 min) with anti-CD14 mAb (undiluted supernatant) or mouse IgG2a (School of Medicine, Rijeka, Croatia) diluted 1:4 in Tris-buffered saline (TBS, 0.05 M Tris containing 0.3 M NaCl; both from Kemika) and 0.1% Tween 20 (Sigma, München, Germany), pH 7.27.6, and followed by incubation (30 min) with the labelled polymer, horseradish peroxidase (HRP) (included in the kit). The initial reaction was completed with 5 min incubation of the liquid 3,3-diaminobenzidine + substrate-chromogen which resulted in a brown-colored precipitate at the antigen site. Incubation with Doublestain block (3 min) removed any cross-reactivity that might be present. The specimens were then incubated (30 min) with the second primary PAM-1 anti-MR mAb at a concentration of 1 µg/100 µl of TBS or mouse IgG1 (Medical School, Rijeka, Croatia) diluted 1:4 in TBS, followed by the incubation of the labelled polymer, AP (included in the kit). The second antigen stain was completed with 5 min incubation with the Fast Red substrate-chromogen which resulted in a red-colored precipitate at the antigen site. Nuclei were counterstained with haematoxylin (Kemika) and specimens were mounted using Liquefy Dako Glycergel (Dako).
Isolation of decidual cells
Decidual tissue was cut into pieces, exposed to collagenase digestion (equal volume of tissue and 0.5% collagenase type IV, Sigma) at 37 °C for 60 min with gentle mixing on the magnetic stirrer. The obtained cell suspension was passed twice through 100 µm nylon mesh (Becton Dickinson, Franklin Lakes, NJ) for tissue debris elimination and centrifuged at 600 g for 10 min. The pellet was resuspended in RPMI 1640 (Institute of Immunology, Zagreb, Croatia), overlaid on Lymphoprep (Nycomed Pharma AS, Oslo, Norway) and centrifuged at 800 g for 20 min. Decidual mononuclear cells (DMC) were collected from the interface, washed twice in RPMI 1640, resuspended in cell culture medium RPMI 1640 supplemented with L-glutamate (2 mM), penicillin (1 x 105 U/l), streptomycin sulphate (0.05 g/l), 10% fetal calf serum (FCS) (all from Gibco, Gaithersburg, MD) and used immediately or cultured overnight (18 h) in 100 x 20 mm tissue culture Petri dishes (TPP, Switzerland) at 37 °C in a humidified atmosphere with 5% CO2.
Immunocytochemistry
Freshly isolated DMC were adjusted to 8 x 105/ml of phosphate-buffered saline (PBS) and 100 µl of the suspension was centrifuged onto a glass microscope slide (500 r.p.m. for 5 min). The cells on the slides were dried at room temperature and fixed in cold acetone. Labeling of the MR was carried out by immunoperoxidase-staining method using a LSABHRP kit (DAKO). Briefly, sections preincubated with LSABHRP kit blocking solution were labeled (30 min) with PAM-1 anti-MR mAb at 1 µg/100 µl in PBS (Na2HPO4.12H2O 33.9 mM, NaCl 136.8 mM, KH2PO4 3 mM of distilled water, all from Kemika)/0.05% bovine serum albumin (BSA, Sigma). Mouse IgG1 supernatant diluted 1:4 in PBS/0.05% BSA was used as isotype matched control. Incubation (10 min) with secondary anti-mouse biotinylated antibodies, was followed by incubation with streptavidin for 10 min (both included in the LSABHRP kit). Reaction was developed using aminoethyl-carbasol (Sigma) and nuclei were counterstained with haematoxylin.
Direct and indirect immunofluorescence
DMC were investigated immediately after isolation or following 18 h culture in the medium only. Some freshly isolated DMC were pretreated with TAG-72 (Sigma) for 30 min on ice (50, 100, 200 or 400 U/ml). One unit (U) is an arbitrary unit related to a reference antigen preparation using the CIS-RIA method. Unconjugated primary PAM-1 anti-MR mAb 1 µg/105 cells/100 µl of fluorescence-activated cell sorter (FACS) buffer NaCl (140 mM), KH2PO4 (1.9 mM), Na2HPO4 (16.5 mM), KCl (3.75 mM) (all from Kemika), Na2EDTA (0.96 mM, Fluka, Buchs, Switzerland), NaN3 (1.5 mM, Difco, Detroit, Michigan, USA), 2% FCS or mouse fluorescein isothiocyanate (FITC)-conjugated IgG1/PE-conjugated IgG2a (Becton-Dickinson, Erembodegen, Belgium) was added to the cells which receptors for Fc fragment of antibodies were blocked. Cells were incubated for 30 min on ice, washed twice in FACS buffer and incubated with FITC-conjugated goat anti-mouse immunoglobulins (Becton-Dickinson) for 30 min on ice. After two washes, PE-conjugated anti-CD14 or PE-conjugated anti-CD83 monoclonal antibody (Becton-Dickinson) was added for 30 min on ice. Following two washes, the pellet was resuspended in 400 µl of 2% paraformaldehyde. Cells were acquired and analyzed in a flow cytometer (Becton Dickinson FACSCalibur) using CellQuestPro software (BD Biosciences, San Jose, California, CA). We acquired 104 DMC in a gate that comprises MR positive events (back gating). The results were expressed as the percentage of MR or mean fluorescence intensity (MFI) for the MR.
Endocytosis
Freshly isolated or 18 h cultured DMC (2 x 105 per sample) were initially incubated 20 min with 10% heat inactivated FCS. The cells were then washed and resuspended in ice-cold medium for endocytosis [Solution A: MgCl2.6H2O 0.5 mM, CaCl2 1.17 mM and Solution B: NaCl 136.8 mM, KCl 2.68 mM, Na2HPO4 7.95 mM, KH2PO4 1.46 mM (all from Kemika) in ratio 1:1, pH 7.4]. Following 10 min incubation on ice the cells were pelleted and treated with TAG-72 (50, 100, 200 or 400 U/ml), PAM-1 anti-MR mAb (10 or 20 µg/ml), mannan (50, 100, 200 or 400 µg/ml, Sigma) or medium in total volume of 100 µl for 30 min on ice. Then ice-cold or warm (37 °C) FITCdextran (Sigma-Aldrich Chemie, Steinheim, Germany) was added to the cells at the final concentration 0.5 mg/ml and the cells were kept on ice or 37 °C for 30 min. Endocytosis was stopped by washing the cells with ice-cold FACS buffer and fixing with 4% paraformaldehyde (10 min at room temperature). After washes, the cells were incubated for 30 min with PE-conjugated anti-CD14 mAb or FITC-conjugated IgG1/PE-conjugated IgG2a, washed twice and resuspend in 2% paraformaldehyde. The cells of CD14 positive phenotype were analyzed in a flow cytometer and data was expressed as MFI for FITCdextran.
Statistical analysis
Non-parametric MannWhitney U test was used to evaluate differences between groups. Differences were considered significant when P<0.05. Data are expressed as mean ± SD. Statistical analysis was carried out with Statistics for Windows, Kernel release 5.5 (StatSoft, Inc., Tulsa, OK).
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Results |
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Discussion |
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The changes in MR expression on decidual macrophages reflected their endocytic activity measured by FITCdextran uptake. FITCdextran is the ligand for the CRD of the MR and is internalized by a receptor-mediated pathway (Kato et al., 2000; East and Isacke, 2002). It is interesting that neither monoclonal PAM-1 anti-MR antibody nor mannan, as the specific ligands for CRD (Chieppa et al., 2003
) even at the highest concentrations used, could completely block FITCdextran uptake by freshly isolated decidual CD14 positive cells, although a statisticaly significant decrease was observed. This is possibly due to the fact that FITCdextran, at a concentration of 0.5 mg/ml enters the cell by an alternative pathwayfluid phase endocytosisthat can not be influenced by binding of the MR (Shurety et al., 1998
). It is also proposed that the alternative pathway of endocytosis increases when the classical receptor mediated endocytosis pathway is somehow disabled (Lamaze and Schmid, 1995
). It is possible that glycoproteins produced within decidual tissue bind to the MR expressing cells, as their natural ligands. Crosslinking of the MR with secretory phase glycoproteins and segregation on the cell membrane may interfere with the internalization routing of a foreign antigen expressed on trophoblast cells, resulting in decreased endocytosis, processing and presentation of trophoblast antigens during the pregnancy. Intimate contact of the MR positive cells with the glandular epithelial cells in early pregnancy indicates their functional interaction. TAG was detected in a variety of human malignancies (Katari et al., 1990
), as well as endometrium of normal female reproductive tract (Osteen et al., 1990
, 1992
). Intense immunostaining in endometrial epithelial cells was limited to the secretory menstrual interval, while TAG-72 was not found in proliferative endometrium (Lessey and Pindzola, 1993
). As TAG-72 appearance correlated with progesterone secretion and accumulation of maternal leukocytes in endometrium (Brackin et al., 2002
), we hypothesized the presence of TAG-72 in the first trimester pregnancy decidua and analyzed its possible binding properties to decidual macrophages. Indeed, TAG-72 significantly diminished PAM-1 anti-MR mAb binding on early decidual CD14 positive cells in a dose dependent manner and reduced FITCdextran uptake by the same cells. It implies that TAG-72 could be the natural ligand for the CRD of the MR in early pregnancy decidua and might be involved in induction of a distinct profile of cytokines and chemokines inefficient in supporting Th1 mediated response, leading to T cell anergy (Chieppa et al., 2003
) specific for the mucosal surfaces, rich in glycoproteins (Akbari et al., 2001
; Weiner, 2001
). Indeed, early decidual macrophages are able to produce various type of cytokines (Kanzaki et al., 1992
; Chaouat et al., 1999; Kitaya et al., 2000
) and to regulate the cytolytic potential of decidual cytolytic T and NK cells (Sotosek et al., 1999
; Laskarin et al., 2002
). However, these cells are hyporeactive to trophoblast alloantigens (Perhar et al., 1989
; Verma et al., 2000
). On the other hand, a few studies indicated that MR participates in cell activation and increasing cytolytic functions (Oshumi and Lee, 1987
; Shibata et al., 1997
). It could be due to activation of the MR with the ligands bound to the epitopes outside CRD, which may be unable to induce anti-inflammatory program, like anti-MR mAb of 19.2 clone in dendritic cells (Chieppa et al., 2003
). A sudden drop in progesterone and diminished glycoprotein production at the end of pregnancy, might enable third trimester decidual macrophages to enhance endocytosis, processing and presentation of the trophoblast antigens by the MR. It might induce Th1 cytokine production and cytotoxic functions of decidual lymphocytes and might promote labour.
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Acknowledgements |
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References |
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Submitted on April 16, 2004; resubmitted on September 29, 2004; accepted on December 13, 2004.
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