Journal of Leukocyte Biology eBioscience full spectrum cell analysis
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published online as doi:10.1189/jlb.0207092 on July 11, 2007

Published online before print July 11, 2007
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0207092v1
82/4/926    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wan, H.
Right arrow Articles by Kiekens, R. C. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wan, H.
Right arrow Articles by Kiekens, R. C. M.
(Journal of Leukocyte Biology. 2007;82:926-933.)
© 2007 by Society for Leukocyte Biology

Chorionic gonadotropin can enhance innate immunity by stimulating macrophage function

Hui Wan1, Marjan A. Versnel, Wai yee Cheung, Pieter J. M. Leenen, Nisar A. Khan, Robbert Benner and Rebecca C. M. Kiekens

Department of Immunology, Erasmus MC, Rotterdam, The Netherlands

1 Correspondence: Department of Immunology, Erasmus MC, Dr. Molewaterplein 50, NL-3015 GE Rotterdam, The Netherlands. E-mail: h.wan{at}erasmusmc.nl


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human chorionic gonadotropin (hCG) is a placental glycoprotein, mainly secreted by trophoblasts during pregnancy. Its function in endocrine regulation has been well documented, but its immunological role is still largely unclear. For a successful pregnancy, an effective innate immunity is needed to protect the mother and fetus against infection, while maintaining tolerance against the paternal antigens of the fetus. The aim of this study was to investigate the effect of hCG on the function of macrophages (M{varphi}), which are major players in the innate response. hCG treatment of IFN-{gamma}-primed M{varphi} resulted in increased production of NO, reactive oxygen species, IL-6 and IL-12p40, and enhanced phagocytosis of apoptotic cells. hCG treatment did not affect the induction of allogeneic T cell proliferation by IFN-{gamma}-primed M{varphi}. The observed effects were receptor-mediated and involved the protein kinase A signaling pathway, as indicated by blocking studies using specific inhibitors. In vivo thioglycollate-elicited M{varphi} also exhibited increased phagocytic ability upon IFN-{gamma} activation and hCG treatment. In conclusion, hCG enhances M{varphi} functions involved in innate immunity, while the capacity to stimulate allogeneic T cells remains unchanged.

Key Words: hCG • M{varphi} • ROS • phagocytosis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Studies about the role of the maternal immune system during pregnancy have focused mainly on the aspect of immune tolerance to the paternal antigens of the fetus. Pregnancy is associated with a systemic shift toward a Th2 cytokine profile, which is characterized by elevated IL-4, IL-5, and IL-10 levels [1 2 3 ]. The observation that many Th1-mediated, autoimmune diseases remit during pregnancy and recur after childbirth might be related to this changed cytokine profile and the hormonal changes that occur during pregnancy [4 ]. A prominent role for the innate immune system in the tolerance to the fetus has been suggested [5 ]. The variety of changes in the immune system led to the concept that the function of the adaptive immune system decreases during pregnancy, and a robust innate immunity is needed to maintain an adequate host defense.

During normal pregnancy, the decidua is populated by multiple leukocytes [6 ]. Cells of the innate immune system, NK cells and macrophages (M{varphi}), are predominant, and the number of lymphocytes is relatively low (1–3%) [7 ]. NK cells produce 90% of pregnancy-induced, uterine IFN-{gamma} [8 ]. M{varphi} constitute 20–30% of the decidual cells at the site of implantation [7 , 9 , 10 ] and unlike NK cells, remain present in high numbers throughout pregnancy [7 , 11 ]. Upon activation by IFN-{gamma}, M{varphi} produce reactive oxygen species (ROS), NO, and proinflammatory cytokines [12 , 13 ]. Oxygen radicals generated from ROS and NO are cytotoxic for a variety of microorganisms including viruses, bacteria, protozoa, and fungi [14 ]. It has been suggested that decidual M{varphi}, using the above-mentioned mechanisms, play an essential role in pathogen recognition, phagocytosis, destruction of microorganisms, and clearance of infected cells during pregnancy to protect the fetus against intrauterine infections [15 ].

Fetal implantation and trophoblast invasion are characterized by a progressive and continuous induction of apoptosis in the maternal tissue surrounding the fetus [16 ]. Resident M{varphi} play an essential role in the removal of apoptotic cells to prevent exposure of the maternal immune system to paternal antigens in the placenta. Furthermore, clearance of apoptotic cells is crucial to the resolution of local inflammation, which is vital in pregnancy [16 ]. Together, this indicates that the innate immune system and the M{varphi} compartment in particular are essential in tolerance toward the fetus and the host protection against infection.

During pregnancy, there are important changes in hormone levels such as the huge production of human chorionic gonadotropin (hCG), which is supposed to influence the immune system. hCG is a placental glycoprotein, also called a pregnancy hormone. The best known function of hCG is to induce the production of progesterone and estrogen during early pregnancy [17 , 18 ]. The biological structure of hCG consists of a unique ß-chain and an {alpha}-chain shared with other glycoprotein hormones, such as the thyroid-stimulating hormone. hCG is secreted mainly by trophoblasts during pregnancy and can be detected in urine and blood. We have reported that hCG and an oligopeptide from the ß-chain of hCG inhibit septic shock in mice [19 ]. The effect of hCG on cells of the immune system is hardly studied. However, this interaction might account for multiple changes in the systemic immune system as well as for the local tolerance induction against the fetus.

It is interesting that M{varphi} in human reproductive tissues, including the decidua and endometria, express luteinizing hormone (LH)/hCG receptors [20 ] and may thus be influenced by the high, local hCG concentration. Binding of hCG to its receptor activates the intracellular cAMP/protein kinase A (PKA) signaling pathway leading to cAMP response element-binding protein (CREB) transcription and contributes to the induction of inducible NO synthase [21 22 23 ]. Indeed, hCG, in combination with IFN-{gamma}, has been found to enhance NO production by murine M{varphi} and microglia [24 , 25 ], but the effect of hCG on other M{varphi} functions is presently unknown. We hypothesize that hCG can promote the innate function of M{varphi} and thereby helps to protect the mother and fetus against infections, while contributing to the maintenance of a successful pregnancy. Therefore, we studied the effect of hCG on oxygen radical induction, cytokine production, and apoptosis of murine bone marrow-derived M{varphi} (BMDM) and peritoneal M{varphi}.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
Specific, pathogen-free C57BL/6 and C3HeB/FeJ female mice were purchased from Harlan (Horst, The Netherlands) and were housed in microisolator cages and given mouse chow and water ad libitum in the animal care facility at Erasmus MC (Rotterdam, The Netherlands). Mice were 8 weeks of age when killed for the isolation of BM cells or subjected to i.p. injection of thioglycollate broth. All experiments were performed with the approval of the Erasmus MC Animal Welfare Committee.

BMDM cultures and activation
BMDM were generated by culturing BM cells from C57BL/6 mice in Petri dishes (Becton Dickinson, Le Pont De Claix, France) at a concentration of 2 x 106 live cells per Petri dish (ø 10 cm) in 10 mL RPMI-1640 culture medium (BioWhittaker, Verviers, Belgium) containing 10% FCS (Hyclone, Perbio, France), 60 mg/mL penicillin and 100 mg/mL streptomycin (BioWhittaker), and 10 ng/mL recombinant mouse (rm)M-CSF (Biosource International, Camarillo, CA, USA) in a 37°C, 5% CO2 incubator. At Day 3, another 10 mL medium with rmM-CSF was added to the cultures.

At Day 5, cells were collected by using a cell scraper (Corning Inc., Corning, NY, USA). For cell activation, BMDM were transferred into 12-well plates (Nunc A/S, Roskilde, Denmark) at a concentration of 1 x 106 cells/well in 1 mL culture medium without rmM-CSF. Cells were stimulated with 50 ng/mL LPS (Sigma Chemical Co., St. Louis, MO, USA), 5 ng/mL rmIFN-{gamma} (Biosource International), according to previous studies [24 , 25 ], 40 ng/mL rmTNF-{alpha} (Biosource International), or PBS. After 6 h, PBS or hCG (Pregnyl, Organon, Oss, The Netherlands) was added at a concentration of 500 U/mL. To inhibit the intracellular PKA signaling pathway, 10 µM H-89 (Biomol, Plymouth Meeting, PA, USA) was added 2 h before the addition of hCG. After 2 days of incubation, supernatants were collected for NO detection and cytokine measurement, and the cells were harvested for phenotyping, ROS detection, and MLR.

Measurement of NO synthesis
NO synthesis in cell cultures was measured using a Griess reagent kit (Molecular Probes, Leiden, The Netherlands) according to the protocols supplied by the manufacturer. In short, 150 µL sample, 20 µL Griess reagent [equal volumes of 0.1% N-(1-naphthyl)-ethylenediamine dihydrochloride and 1% sulfanilic acid], and 130 µL deionized water were mixed and incubated at room temperature for 30 min. Sodium nitrite (1–100 µM) was used as standard, and the absorbance was measured at 540 nm by an ELISA reader (Thermo Labsystems, Helsinki, Finland).

Measurement of ROS
ROS production was measured by labeling BMDM with 5-(and 6-) chloromethyl-2',7'-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA; Molecular Probes). Briefly, 1 x 106 cells were resuspended in 1 mL-prewarmed PBS, and then 1 µL CM-H2DCFDA (5 µg/mL) was added, and cells were incubated for 10 min at 37°C. Upon reaction of CM-H2DCFDA with ROS, a fluorescent product is generated. Fluorescence was detected and analyzed on a FACSCalibur flow cytometer using CellQuest analysis software (Becton Dickinson).

Flow cytometry
At Day 7, BMDM were collected and labeled with the following antibodies: ER-TR3-bio [MHC class II (MHCII), BMA, Augst, Switzerland], streptavidin-APC, CD11b-FITC, CD40-PE, IgG control (all from BD PharMingen, Erembodegem-Aalst, Belgium), and F4/80-FITC (Caltag, South San Francisco, CA, USA); they were analyzed using FACSCalibur (Becton Dickinson). In between incubations, cells were washed twice with PBS + BSA (5%, Celliance Corp., Atlanta, GA, USA). All steps were performed at room temperature. Data were analyzed by WinMDI 2.8 software and depicted by mean fluorescence intensity (MFI) and percentage of positive cells.

Cytokine detection
ELISA kits for the detection of murine TNF-{alpha}, IL-6, IL-10 (Biosource International), and IL-12p40 (R&D Systems, Oxen, UK) were used according to the protocols supplied by the manufacturer. In short, plates were coated with capture antibody for 18 h at 4°C and washed with PBS-Tween (0.05%). Diluted culture supernatants and standards were added and incubated for 2 h at room temperature. Thereafter, the biotin-labeled detection antibody was added, followed by incubation with streptavidin-HRP for 30 min, chromogen tetramethylbenzidine was added for 30 min, followed by the addition of the stop solution. In between incubations, the plates were washed with PBS-0.05% Tween. The absorbance was measured at 450 or 450 and 650 nm by an ELISA reader (Thermo Labsystems).

T cell proliferation
Allogeneic MLR were performed in round-bottom, 96-microwell plates (Nunc A/S). Naïve T cells were obtained from spleens of C3HeB/FeJ mice, after lysis of the RBCs by Gey's medium (Millipore, Billerica, MA, USA) and negative depletion by incubation with a combination of antibodies: CD11b (M1/70), CD45R (B220), and MHCII (M5/114; American Type Culture Collection, Manassas, VA, USA) and anti-rat IgG microbeads (Miltenyi Biotec, Auburn, CA, USA), followed by Automacs separation (Miltenyi Biotec). BMDM were added in various concentrations to T cells (1.5x106 cells/well) and cocultured for 3 days in RPMI-1640 medium containing 10% FCS, 60 mg/mL penicillin, and 100 mg/mL streptomycin. Proliferation of T cells was measured by uptake of 3H-thymidine (1 µCi/well, DuPont-NEN, Boston, MA, USA) over a period of 16 h and expressed as cpm.

In vitro phagocytosis of microspheres and apoptotic cells
BMDM were incubated with FluoSpheres® polystyrene microspheres (1.0 µm, yellow-green fluorescent, Molecular Probes) to measure the extent of phagocytosis. In each tube, 106 cells were mixed with 40 x 106 beads. After 2 h incubation at 37°C and in 5% CO2, cells were washed with PBS, and then fluorescence was measured using flow cytometry. Another portion of the cells was prepared for immunofluorescence microscopy. Cytospins were prepared, air-dried, and fixed in acetone for 5 min. After blocking with 1% BSA, the slides were incubated with BM8-biotin, streptavidin-Texas Red (Caltag), and 4,6-diamidino-2-phenylindole (DAPI; detection of DNA content, Vector Laboratories Inc., Burlingame, CA, USA) sequentially, prior to analysis using a fluorescence microscope.

Apoptosis was induced in human Jurkat T cells by exposure to UV radiation (UVB312 nm, equivalent to 800 mJ/cm2), followed by 24 h culture in RPMI-1640 medium containing 10% FCS, 60 mg/mL penicillin, and 100 mg/mL streptomycin [26 ]. This resulted in a population of cells, which was ~50% apoptotic, as determined by Annexin V and propidium iodide staining (Calbiochem, Darmstadt, Germany). To label apoptotic cells, 5 µM CFSE (Molecular Probes) was added to 107 cells/mL and incubated for 10 min on melting ice.

For the collection of in vivo-conditioned peritoneal exudates, C57BL/6 mice were injected i.p. with hCG (300 U/20 g body weight) or PBS in a volume of 200 µL. One hour later, 1 mL sterile 4% Brewer's thioglycollate broth (Sigma-Aldrich, Poole, UK) was injected i.p. per mouse; PBS was used as a control. At Day 3, the mice were killed, and the peritoneum was flushed with 4 mL PBS and 1 mL air to harvest cells. A portion of the collected peritoneal cells was incubated with CFSE-labeled, apoptotic cells (1:5) for 30 min at 37°C, followed by BM8 staining.

The rest of the cells were cultured in vitro in RPMI-1640 medium containing 10% FCS, 60 mg/mL penicillin, and 100 mg/mL streptomycin with 5 ng/mL IFN-{gamma}, with or without 500 U/mL hCG for 2 days. The cells were collected and fed with CFSE-labeled, apoptotic cells for 30 min at 37°C, followed by BM8 staining. Using flow cytometry, the percentage of double-positive cells representing M{varphi}, which have ingested apoptotic cells, so-called phagocytic M{varphi}, was determined. Phagocytosis of apoptotic cells by M{varphi} was also analyzed morphologically on cytospins, where cells were stained with Diff-Quick (Dade Diff-Quick, Düdingen, Switzerland).

Statistical analyses
Data are expressed as mean values ± SEM in all figures. All statistical analyses were performed using Student's t-test or paired t-test. P values <0.05 were considered significant: *, P < 0.05; **, P < 0.01; ***, P < 0.001.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
hCG treatment enhances IFN-{gamma}-induced NO production by BMDM
LPS and IFN-{gamma} but not TNF-{alpha} increased NO production significantly by BMDM (Fig. 1 ). hCG alone did not influence NO synthesis by BMDM, but the addition of hCG to IFN-{gamma}-primed M{varphi} enhanced NO production significantly. hCG did not influence NO production by LPS or TNF-{alpha}-activated BMDM (Fig. 1) .


Figure 1
View larger version (21K):
[in this window]
[in a new window]

  		Figure 1. Increased NO production by IFN-{gamma}-primed BMDM upon hCG treatment. BM cells were cultured for 5 days in the presence of M-CSF. Cells were collected and stimulated with the following stimuli: PBS, TNF-{alpha}, LPS, or IFN-{gamma}. Six hours later, hCG or PBS was added to each culture. After 2 days of culture, NO production in the culture supernatants was measured.

 
hCG increases IFN-{gamma}-induced ROS production by BMDM
BMDM were stimulated with IFN-{gamma} in the presence or absence of hCG to investigate the effect of hCG on the production of ROS, determined by fluorescent visualization using CM-H2DCFDA, which upon cleavage and subsequent oxidation by ROS, yields a fluorescent product. Flow cytometry revealed two populations: ROS-positive cells (Gates R1 and R2) and ROS-negative cells (lower part; Fig. 2A ). Activation of BMDM with IFN-{gamma} increased the percentage of ROS-positive cells (Fig. 2D) . hCG treatment of IFN-{gamma}-primed BMDM with hCG increased ROS production significantly in a dose- and time-dependent manner (Fig. 2B and data not shown). ROS production increased steadily in time and reached a maximum after 48 h of incubation (data not shown). In all cases, the percentage of ROS-positive cells gave similar results as the MFI. Differences in pH of the incubation media of stimulated cells were not found at the moment of cell harvest, thus excluding the pH as the cause of the observed differences in ROS production (data not shown).


Figure 2
View larger version (47K):
[in this window]
[in a new window]

  		Figure 2. hCG increases receptor-mediated ROS production by IFN-{gamma}-primed BMDM. Cultured BMDM were stimulated with IFN-{gamma}. Six hours later, hCG (0, 0.5, 5, 50, 500 U/mL) or PBS was added. After 2 days of culture, ROS production was determined. The CM-H2DCFDA-positive cells (R2) in the dot-plot (A) were ROS-producing M{varphi}. SSC, Side-scatter; FSC, forward-scatter. (B) Dose-dependent ROS production by IFN-{gamma}-primed BMDM upon hCG treatment. A representative of three individual experiments is shown. (C and D) Increased ROS production by IFN-{gamma}-primed BMDM upon hCG treatment was mediated via the PKA signaling pathway. BMDM were primed with IFN-{gamma}; after 4 h, the PKA inhibitor (H-89, 10 µM) was added, and 2 h later, hCG (500 U/mL) or PBS was added. Addition of the PKA inhibitor H-89 blocked the increased ROS production by IFN-{gamma}-primed BMDM upon hCG treatment. The MFI and percentage of ROS-positive cells from three to six separate experiments are depicted.

 
hCG binding to its receptor activates the intracellular cAMP/PKA signaling pathway [21 , 22 ]. To study whether the increased ROS production induced by hCG involves this pathway, the PKA inhibitor H-89 was added 2 h prior to the addition of hCG. H-89 reduced the increased ROS production by IFN-{gamma}-primed BMDM significantly upon hCG treatment (Fig. 2C and 2D) in a dose-dependent manner (data not shown).

hCG increases cytokine production by IFN-{gamma}-primed BMDM
Unstimulated BMDM secreted small amounts of IL-6, IL-10, and IL-12p40. Activation of BMDM by a low concentration of IFN-{gamma} (5 ng/mL) did not increase cytokine production (Fig. 3 ). The addition of an increasing amount of hCG to IFN-{gamma}-primed BMDM caused a dose-dependent increase in IL-6 and IL-12p40 production. IL-10 levels remained unaltered up to 500 U/mL hCG. To characterize further which form of IL-12p40 was induced, IL-12p40 monomer, homodimer (IL-12p80), heterodimer (IL-12p70), and IL-23 concentrations were determined. IL-12p40 monomer and homodimer were increased significantly by IFN-{gamma}-primed BMDM upon hCG treatment, and the levels of IL-12p70 and IL-23 were low and remained unchanged after addition of hCG (data not shown).


Figure 3
View larger version (10K):
[in this window]
[in a new window]

  		Figure 3. Cytokine production by IFN-{gamma}-primed BMDM upon hCG treatment. BMDM were primed with IFN-{gamma}, and after 6 h, hCG (0, 0.5, 5, 50, or 500 U/mL) or PBS was added. After 2 days of culture, the culture supernatants were analyzed for IL-6, IL-12p40, and IL-10 by ELISA. The results shown are representative from three independent experiments.

 
We excluded the possible LPS contamination in hCG by showing unchanged TNF-{alpha} production by IFN-{gamma}-primed BMDM (data not shown) and insensitivity to polymyxin B, an agent that neutralizes the effects of LPS (data not shown).

hCG treatment does not change the BMDM phenotype and their ability to induce allogeneic T cell proliferation
Treatment of BMDM with IFN-{gamma} increased CD11b, CD40, and MHCII membrane expression, but the expression of F4/80 remained unchanged. The addition of hCG to IFN-{gamma}-primed BMDM did not change the expression levels of these markers (Fig. 4A ).


Figure 4
View larger version (22K):
[in this window]
[in a new window]

  		Figure 4. Phenotypic changes and allogeneic T cell proliferation by IFN-{gamma}-primed BMDM upon hCG treatment. BMDM were stimulated with IFN-{gamma}. After 6 h, hCG (500 U/mL) or PBS was added. (A) After 2 days of culture, M{varphi} were collected and labeled for F4/80, CD11b, MHCII, and CD40. The expression is depicted as MFI. (B) M{varphi} were cocultured with allogeneic spleen T cells. After coculturing M{varphi} and T cells (ratio 1:5) for 3 days, proliferation of T cells was measured by uptake of 3H-thymidine. LPS-activated DC were used as a positive control, which gave approximately 50,000 cpm after coculture with T cells. The data are pooled from four separate experiments.

 
Unstimulated BMDM induced allogeneic T cell proliferation only to a limited extent. Addition of IFN-{gamma} at low concentration or hCG showed a trend toward an increased T cell proliferation. hCG treatment of IFN-{gamma}-primed BMDM also did not change their ability to stimulate T cell proliferation (Fig. 4B) . LPS-activated DC were used as a positive control.

hCG increases the phagocytic capacity of IFN-{gamma}-primed BMDM
BMDM, identified by the expression of the M{varphi} marker BM8/F4/80, readily phagocytosed FluoSpheres® polystyrene microspheres (Fig. 5A ). Flow cytometry enabled convenient quantification of fluorescence-positive cells, which have phagocytosed variable amounts of labeled microspheres (Fig. 5B) . IFN-{gamma} priming of BMDM resulted in a 10% increase in cells that phagocytosed beads compared with unstimulated BMDM. Treatment of IFN-{gamma}-primed BMDM with hCG resulted in a further 20% increase of phagocytic cells. The amount of microspheres, which were phagocytosed by a single cell, was also increased in hCG-treated cells compared with controls. The effect of hCG could be blocked by addition of the PKA signaling pathway inhibitor H-89, suggesting hCG receptor involvement (Fig. 5C) .


Figure 5
View larger version (90K):
[in this window]
[in a new window]

  		Figure 5. Increased phagocytosis by IFN-{gamma}-primed BMDM and peritoneal M{varphi} upon hCG treatment. BMDM were stimulated with IFN-{gamma}. After 6 h, hCG (500 U/mL) or PBS was added. After 2 days of culture, the cells were collected and incubated with fluorescence-labeled, polystyrene microspheres for 30 min. Subsequently, the cells were prepared for immunofluorescence staining on cytospin A. Red, BM8; green, fluorescence beads; blue, DAPI; left with DAPI; right without DAPI. (B) Flow cytometric analysis of phagocytosis of fluorescence-labeled microspheres. The multiple lines in the figure represent cells that phagocytosed different amounts of beads. The result is representative from three separate experiments. (C) Proportion of phagocytic cells upon IFN-{gamma} priming, hCG stimulation, and inhibition of PKA signaling by H-89. Data are pooled from three separate experiments. C57BL/6 mice were injected i.p. with hCG or PBS. One hour later, thioglycollate broth was injected i.p. On Day 3, mice were killed, and peritoneal cells were collected. Part of the cells was incubated with CFSE-labeled apoptotic T cells, followed by BM8 staining; the other cells were cultured in vitro with PBS, IFN-{gamma} (5 ng/mL), or IFN-{gamma} plus hCG (5 ng/mL+500 U/mL). After 2 days, cells were collected and incubated with CFSE-labeled, apoptotic T cells, followed by BM8 staining. Phagocytosis was recorded on cytospins using Diff-Quick staining (D). The peritoneal cells appear large and light blue with diffuse edges; the early apoptotic cells were small and round and appeared dark blue/purple; late apoptotic cells were even smaller and showed shrunken and segmented nuclei; the color was completely pink. The continuous process of phagocytosis of apoptotic cells was recorded as follows: I, Nonapoptotic T cells; II, nonphagocytic M{varphi} and early stage of apoptotic T cells (arrows); III, late stage of apoptotic T cells (arrows) and phagocytic M{varphi} with protruded arms, which encircled the apoptotic T cells (arrows); IV, apoptotic T cell phagocytosed completely by a M{varphi} (arrow). (E) In flow cytometry, the percentage of BM8+CFSE+ double-positive cells was determined, representing the phagocytic M{varphi} (n=5).

 
hCG increases phagocytosis of apoptotic cells by thioglycollate-activated, peritoneal M{varphi}
To investigate whether stimulation of phagocytosis by hCG was a general phenomenon, we also approached this in an alternative setting. Mice were pretreated with hCG i.p. and 1 h later, injected with thioglycollate to induce peritonitis. On Day 3, peritoneal cells were collected; the majority of these cells were M{varphi} (60–80%), as determined by flow cytometry. By using the method of subset analysis described previously [27 ], hCG did not influence the cellular composition of the infiltrate isolated. Peritoneal cells were incubated with CFSE-labeled apoptotic T cells and analyzed directly or were cultured first with IFN-{gamma}, with or without hCG for 2 days prior to analyzing the phagocytic capacity. In Figure 5D , four sequential stages in the phagocytic process of apoptotic cells are depicted (I–IV). From stage IV, M{varphi} were counted as phagocytic cells. The percentage of BM8+CFSE+ double-positive cells was determined, representing M{varphi}, which have ingested apoptotic cells (Fig. 5E) . A slightly higher frequency of thioglycollate-recruited M{varphi} phagocytosed apoptotic cells compared with resident peritoneal cells. Without thioglycollate injection in vivo, treatment of hCG did not change the phagocytic ability of peritoneal cells. However, ex vivo culture with IFN-{gamma} increased their phagocytic ability significantly. Ex vivo culture of peritoneal M{varphi} recruited by thioglycollate injection with IFN-{gamma} resulted in an increased phagocytic capacity, and ex vivo culture with IFN-{gamma} and hCG increased the phagocytic capacity further compared with IFN-{gamma} culture alone. Moreover, thioglycollate-induced peritoneal cells, after in vivo treatment with hCG, followed by ex vivo stimulation with IFN-{gamma} in the presence of hCG, revealed a higher phagocytic capacity, indicating that continuous presence of hCG increased phagocytic capacity further.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
To test our hypothesis that hCG stimulates innate immunity to protect mother and fetus against infection, we studied the effect of hCG on innate immune functions of BMDM and inflammatory peritoneal M{varphi}. In general, M{varphi} play an essential role in innate immunity as well as adaptive immunity, by exerting specific functions, such as removal of debris, dead cells, and microorganisms and the production of oxygen radicals and cytokines.

In agreement with previous studies about peritoneal M{varphi} and microglia [24 , 25 ], we found that hCG alone did not influence NO synthesis, and activation by IFN-{gamma} did increase NO production by BMDM. However, hCG further increased NO and ROS production significantly by BMDM treated with IFN-{gamma}. Radicals generated from NO and ROS are toxic for a variety of microorganisms, including viruses. Thus, increased NO and ROS production enhances the defensive capacity against invading pathogens [14 ].

M{varphi} produce proinflammatory cytokines upon activation [13 ]. hCG increased IL-6 and IL-12 mRNA (data not shown) and protein production significantly by IFN-{gamma}-primed BMDM, while IL-10 and TNF-{alpha} levels did not change. IL-6 is considered to be a proinflammatory cytokine, and it also promotes Th2 differentiation from Th0 cells and inhibits Th1 polarization simultaneously [28 ]. In addition, IL-6 plays a crucial, anti-inflammatory role in local and systemic acute inflammatory responses by down-regulating TNF-{alpha} and MIP-2 but not the anti-inflammatory cytokine IL-10 [29 ]. The survival rate of IL-6 knockout mice during endotoxemia was ~50% lower than of IL-6 wild-type mice [29 ], indicating a protective effect of IL-6 under these conditions. Thus, by increasing IL-6 production, hCG can contribute to the Th2 environment during pregnancy as well as enhanced protection against inflammation.

IL-12p40 expression was also increased by hCG. However, no effect was seen on IL-12p70 and IL-23 production. IL-12 exists in vivo in three forms: IL-12p70 heterodimer, consisting of IL-12p40 and p35, IL-12p40 homodimer, and IL-12p40 monomer [30 ]. M{varphi} are major producers of IL-12 and secrete relatively more p40 than p35 subunits. IL-12p70 plays a key role in the differentiation of Th1 cells and resistance to bacterial infections. Conversely, homodimeric IL-12p40 can compete with the heterodimeric p70 for binding to the high-affinity IL-12R, thus inhibiting IL-12p70 activity [31 32 33 34 35 36 ]. Mice lacking p40 have a higher mortality rate and bacterial burden when infected with Salmonella, compared with mice, which were capable of producing p40 but were p35-deficient [33 , 37 ]. Similarly, humans deficient for p40 have a hampered ability to fight infections of Bacille Calmette-Guerin and Salmonella and undergo recurrent episodes of pneumonia with sepsis [38 , 39 ]. These data indicate that IL-12p40 is a necessary component of human immunity against bacteria. The observed increase of IL-12p40 by M{varphi} upon hCG treatment may therefore enhance the innate immune response during pregnancy.

Blocking of the cAMP/PKA signaling pathway, which is induced upon hCG receptor activation, reduced the increased ROS production by IFN-{gamma}-primed BMDM upon hCG treatment significantly. This indicates that the observed effect of hCG on ROS production by M{varphi} probably involves receptor mediation. Binding of hCG to hCG/LH receptor activates the intracellular cAMP/PKA signaling pathway leading to CREB transcription and inhibition of the TNF signaling pathway [21 , 22 ]. It has also been shown that activation of CREB leads to the induction of NO, IL-6, and TNF-{alpha} [23 , 40 , 41 ]. In our model, the TNF-{alpha} production remained unchanged, which may be a result of the simultaneous inhibition and induction of TNF-{alpha} production by hCG. Inhibition of the PKA signaling pathway did not reduce IL-6 and IL-12p40 protein production by IFN-{gamma}-primed BMDM upon hCG treatment (data not shown), which indicates a separate pathway.

IFN-{gamma}-primed BMDM appeared functional-activated upon hCG treatment, indicated by the increased production of NO, ROS, IL-6, and IL-12p40. However, the expression of F4/80, CD11b, MHCII, and CD40 did not alter. Also, the capacity of hCG-treated, IFN-{gamma}-primed BMDM to induce allogeneic T cell proliferation was unchanged. This suggests that hCG did not affect the capacity of M{varphi} to stimulate adaptive immunity via T cell activation.

A major function of M{varphi} is to phagocytose apoptotic cells and harmful particles and to take up and destroy microorganisms. Simultaneously, hazardous, inflammatory, immune reactions should be inhibited. Upon IFN-{gamma} priming and hCG treatment, a higher percentage of M{varphi} became phagocytic. These phagocytic cells showed an increased phagocytosis of beads. Blocking of the cAMP/PKA signaling pathway reduced this effect significantly, indicating receptor involvement in the hCG effect. Recruited inflammatory peritoneal M{varphi}, which were isolated after in vivo treatment with hCG, did not exhibit a change in phagocytic capability. However, further ex vivo stimulation with IFN-{gamma} in the continuous presence of hCG did cause an increased phagocytosis, emphasizing the additional stimulating capacity of hCG on the IFN-{gamma}-induced increase of phagocytic capacity. There are several possible explanations for this observation: In vivo thioglycollate stimulation and in vitro IFN-{gamma} activation provide different inflammatory signals to hCG-treated M{varphi}; inflammatory-recruited M{varphi}, but not resident peritoneal cells, are more comparable with BMDM; hCG alone cannot affect phagocytosis, but an additional inflammatory signal is required for the newly thioglycollate-recruited M{varphi} to exert the effect. Another difference could be the different hCG concentration between in vitro and in vivo conditions. For our in vivo experiments, we chose the effective dose used in other in vivo experiments, revealing the influence of hCG on the immune system [19 , 42 , 43 ]. Our data indicate that the continuous presence of hCG and an activator such as IFN-{gamma} are required for the increased phagocytosis by M{varphi} in our model. hCG is produced in high amounts by trophoblast cells, especially in the first trimester of pregnancy. Simultaneously, apoptosis is prominent in the trophoblast layer of the placenta, suggesting that cell turnover at the site of implantation is necessary for the appropriate growth and function of the placenta [44 , 45 ].

It should be noted that purified hCG preparations contain various breakdown products, including peptides and hCG-associated fragments, many of which are identified [19 , 46 ]. Although we used clinical-grade hCG, and our data suggested the whole molecule instead of the breakdown products that exert the observed effect, the function of individual peptides needs to be investigated further.

In conclusion, hCG can promote innate functions of M{varphi}, such as the clearance of apoptotic cells and the resolution of inflammation, which are highly relevant for the maintenance of pregnancy.


   ACKNOWLEDGEMENTS
 
This study was financially supported by Biotempt B.V., Koekange, The Netherlands. We thank Tar van Os for his support in preparation of the figures, Dr. Milton A. P. Oliveira and Dr. Manon E. Wildenberg for their helpful discussion and suggestions, and Cornelia G. van Helden-Meeuwsen and Jojanneke M. C. Coppens for their technical support.

Received February 6, 2007; revised May 10, 2007; accepted May 31, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Weetman, A. P. (1999) The immunology of pregnancy Thyroid 9,643-646[Medline]
  2. Saito, S. (2000) Cytokine network at the feto-maternal interface J. Reprod. Immunol. 47,87-103[Medline]
  3. Clark, D. A., Chaouat, G., Arck, P. C., Mittruecker, H. W., Levy, G. A. (1998) Cytokine-dependent abortion in CBA x DBA/2 mice is mediated by the procoagulant fgl2 prothrombinase J. Immunol. 160,545-549[Abstract/Free Full Text]
  4. Wilder, R. L. (1998) Hormones, pregnancy, and autoimmune diseases Ann. N. Y. Acad. Sci. 840,45-50[CrossRef][Medline]
  5. Sacks, G., Sargent, I., Redman, C. (1999) An innate view of human pregnancy Immunol. Today 20,114-118[CrossRef][Medline]
  6. Mincheva-Nilsson, L., Baranov, V., Yeung, M. M., Hammarstrom, S., Hammarstrom, M. L. (1994) Immunomorphologic studies of human decidua-associated lymphoid cells in normal early pregnancy J. Immunol. 152,2020-2032[Abstract]
  7. Lessin, D. L., Hunt, J. S., King, C. R., Wood, G. W. (1988) Antigen expression by cells near the maternal-fetal interface Am. J. Reprod. Immunol. Microbiol. 16,1-7[Medline]
  8. Ashkar, A. A., Croy, B. A. (2001) Functions of uterine natural killer cells are mediated by interferon {gamma} production during murine pregnancy Semin. Immunol. 13,235-241[CrossRef][Medline]
  9. Kabawat, S. E., Mostoufi-Zadeh, M., Driscoll, S. G., Bhan, A. K. (1985) Implantation site in normal pregnancy. A study with monoclonal antibodies Am. J. Pathol. 118,76-84[Abstract]
  10. Uckan, D., Steele, A., Cherry, , Wang, B.Y., Chamizo, W., Koutsonikolis, A., Gilbert-Barness, E., Good, R. A. (1997) Trophoblasts express Fas ligand: a proposed mechanism for immune privilege in placenta and maternal invasion Mol. Hum. Reprod. 3,655-662[Abstract/Free Full Text]
  11. Khong, T. Y. (1987) Immunohistologic study of the leukocytic infiltrate in maternal uterine tissues in normal and preeclamptic pregnancies at term Am. J. Reprod. Immunol. Microbiol. 15,1-8[Medline]
  12. Taylor, P. R., Martinez-Pomares, L., Stacey, M., Lin, H. H., Brown, G. D., Gordon, S. (2005) Macrophage receptors and immune recognition Annu. Rev. Immunol. 23,901-944[CrossRef][Medline]
  13. Gordon, S. (2003) Alternative activation of macrophages Nat. Rev. Immunol. 3,23-35[CrossRef][Medline]
  14. Vazquez-Torres, A., Jones-Carson, J., Baumler, A. J., Falkow, S., Valdivia, R., Brown, W., Le, M., Berggren, R., Parks, W. T., Fang, F. C. (1999) Extraintestinal dissemination of Salmonella by CD18-expressing phagocytes Nature 401,804-808[CrossRef][Medline]
  15. Singh, U., Nicholson, G., Urban, B. C., Sargent, I. L., Kishore, U., Bernal, A. L. (2005) Immunological properties of human decidual macrophages—a possible role in intrauterine immunity Reproduction 129,631-637[Abstract/Free Full Text]
  16. Mor, G., Abrahams, V. M. (2003) Potential role of macrophages as immunoregulators of pregnancy Reprod. Biol. Endocrinol. 1,119[CrossRef][Medline]
  17. Pierce, J. G., Parsons, T. F. (1981) Glycoprotein hormones: structure and function Annu. Rev. Biochem. 50,465-495[CrossRef][Medline]
  18. Keay, S. D., Vatish, M., Karteris, E., Hillhouse, E. W., Randeva, H. S. (2004) The role of hCG in reproductive medicine BJOG 111,1218-1228[CrossRef][Medline]
  19. Khan, N. A., Khan, A., Savelkoul, H. F., Benner, R. (2002) Inhibition of septic shock in mice by an oligopeptide from the ß-chain of human chorionic gonadotrophin hormone Hum. Immunol. 63,1-7[CrossRef][Medline]
  20. Zhang, Y. M., Rao Ch, V., Lei, Z. M. (2003) Macrophages in human reproductive tissues contain luteinizing hormone/chorionic gonadotropin receptors Am. J. Reprod. Immunol. 49,93-100[CrossRef][Medline]
  21. Rao, ChV, Li, X., Manna, S. K., Lei, Z. M., Aggarwal, B. B. (2004) Human chorionic gonadotropin decreases proliferation and invasion of breast cancer MCF-7 cells by inhibiting NF-{kappa}B and AP-1 activation J. Biol. Chem. 279,25503-25510[Abstract/Free Full Text]
  22. Manna, S. K., Mukhopadhyay, A., Aggarwal, B. B. (2000) Human chorionic gonadotropin suppresses activation of nuclear transcription factor-{kappa} B and activator protein-1 induced by tumor necrosis factor J. Biol. Chem. 275,13307-13314[Abstract/Free Full Text]
  23. Chio, C. C., Chang, Y. H., Hsu, Y. W., Chi, K. H., Lin, W. W. (2004) PKA-dependent activation of PKC, p38 MAPK and IKK in macrophage: implication in the induction of inducible nitric oxide synthase and interleukin-6 by dibutyryl cAMP Cell. Signal. 16,565-575[CrossRef][Medline]
  24. Kim, H. M., Moon, Y. H. (1996) Human chorionic gonadotropin induces nitric oxide synthase mRNA in mouse peritoneal macrophages Biochem. Biophys. Res. Commun. 229,548-552[CrossRef][Medline]
  25. Kim, H. M., Rim, H. K., Shin, T., Kim, J. J., Park, S. T., Oh, J. M., Choi, M. K., Chung, Y. T., Rhee, H. S., Jeung, J. Y., Lee, K. N., Kim, N. S., Kim, C. H. (2000) Human chorionic gonadotropin induces nitric oxide synthesis by murine microglia Int. J. Immunopharmacol. 22,453-461[CrossRef][Medline]
  26. Norsworthy, P. J., Fossati-Jimack, L., Cortes-Hernandez, J., Taylor, P. R., Bygrave, A. E., Thompson, R. D., Nourshargh, S., Walport, M. J., Botto, M. (2004) Murine CD93 (C1qRp) contributes to the removal of apoptotic cells in vivo but is not required for C1q-mediated enhancement of phagocytosis J. Immunol. 172,3406-3414[Abstract/Free Full Text]
  27. Bouma, G., Nikolic, T., Coppens, J. M., van Helden-Meeuwsen, C. G., Leenen, P. J., Drexhage, H. A., Sozzani, S., Versnel, M. A. (2005) NOD mice have a severely impaired ability to recruit leukocytes into sites of inflammation Eur. J. Immunol. 35,225-235[CrossRef][Medline]
  28. Diehl, S., Rincon, M. (2002) The two faces of IL-6 on Th1/Th2 differentiation Mol. Immunol. 39,531-536[CrossRef][Medline]
  29. Xing, Z., Gauldie, J., Cox, G., Baumann, H., Jordana, M., Lei, X. F., Achong, M. K. (1998) IL-6 is an antiinflammatory cytokine required for controlling local or systemic acute inflammatory responses J. Clin. Invest. 101,311-320[Medline]
  30. Cooper, A. M., Khader, S. A. (2007) IL-12p40: an inherently agonistic cytokine Trends Immunol. 28,33-38[CrossRef][Medline]
  31. Trinchieri, G. (1998) Interleukin-12: a cytokine at the interface of inflammation and immunity Adv. Immunol. 70,83-243[Medline]
  32. Abdi, K. (2002) IL-12: the role of p40 versus p75 Scand. J. Immunol. 56,1-11[CrossRef][Medline]
  33. Gazzinelli, R. T., Talvani, A., Camargo, M. M., Santiago, H. C., Oliveira, M. A., Vieira, L. Q., Martins, G. A., Aliberti, J. C., Silva, J. S. (1998) Induction of cell-mediated immunity during early stages of infection with intracellular protozoa Braz. J. Med. Biol. Res. 31,89-104[Medline]
  34. Oliveira, M. A., Lima, G. M., Shio, M. T., Leenen, P. J., Abrahamsohn, I. A. (2003) Immature macrophages derived from mouse bone marrow produce large amounts of IL-12p40 after LPS stimulation J. Leukoc. Biol. 74,857-867[Abstract/Free Full Text]
  35. Oliveira, M. A., Tadokoro, C. E., Lima, G. M., Mosca, T., Vieira, L. Q., Leenen, P. J., Abrahamsohn, I. A. (2005) Macrophages at intermediate stage of maturation produce high levels of IL-12 p40 upon stimulation with Leishmania Microbes Infect 7,213-223[CrossRef][Medline]
  36. Trinchieri, G. (2003) Interleukin-12 and the regulation of innate resistance and adaptive immunity Nat. Rev. Immunol. 3,133-146[CrossRef][Medline]
  37. Lehmann, J., Bellmann, S., Werner, C., Schroder, R., Schutze, N., Alber, G. (2001) IL-12p40-dependent agonistic effects on the development of protective innate and adaptive immunity against Salmonella enteritidis J. Immunol. 167,5304-5315[Abstract/Free Full Text]
  38. Altare, F., Lammas, D., Revy, P., Jouanguy, E., Doffinger, R., Lamhamedi, S., Drysdale, P., Scheel-Toellner, D., Girdlestone, J., Darbyshire, P., Wadhwa, M., Dockrell, H., Salmon, M., Fischer, A., Durandy, A., Casanova, J. L., Kumararatne, D. S. (1998) Inherited interleukin 12 deficiency in a child with bacille Calmette-Guerin and Salmonella enteritidis disseminated infection J. Clin. Invest. 102,2035-2040[Medline]
  39. Haraguchi, S., Day, N. K., Nelson, R. P., Jr, Emmanuel, P., Duplantier, J. E., Christodoulou, C. S., Good, R. A. (1998) Interleukin 12 deficiency associated with recurrent infections Proc. Natl. Acad. Sci. USA 95,13125-13129[Abstract/Free Full Text]
  40. Grassl, C., Luckow, B., Schlondorff, D., Dendorfer, U. (1999) Transcriptional regulation of the interleukin-6 gene in mesangial cells J. Am. Soc. Nephrol. 10,1466-1477[Abstract/Free Full Text]
  41. Roach, S. K., Lee, S. B., Schorey, J. S. (2005) Differential activation of the transcription factor cyclic AMP response element binding protein (CREB) in macrophages following infection with pathogenic and nonpathogenic mycobacteria and role for CREB in tumor necrosis factor {alpha} production Infect. Immun. 73,514-522[Abstract/Free Full Text]
  42. Khan, N. A., Khan, A., Savelkoul, H. F., Benner, R. (2001) Inhibition of diabetes in NOD mice by human pregnancy factor Hum. Immunol. 62,1315-1323[CrossRef][Medline]
  43. Benner, R., Khan, N. A. (2005) Dissection of systems, cell populations and molecules Scand. J. Immunol. 62(Suppl. 1),62-66[CrossRef][Medline]
  44. Ratts, V. S., Tao, X. J., Webster, C. B., Swanson, P. E., Smith, S. D., Brownbill, P., Krajewski, S., Reed, J. C., Tilly, J. L., Nelson, D. M. (2000) Expression of BCL-2, BAX and BAK in the trophoblast layer of the term human placenta: a unique model of apoptosis within a syncytium Placenta 21,361-366[CrossRef][Medline]
  45. Levy, R., Smith, S. D., Yusuf, K., Huettner, P. C., Kraus, F. T., Sadovsky, Y., Nelson, D. M. (2002) Trophoblast apoptosis from pregnancies complicated by fetal growth restriction is associated with enhanced p53 expression Am. J. Obstet. Gynecol. 186,1056-1061[CrossRef][Medline]
  46. Bartocci, A., Welker, R. D., Schlick, E., Chirigos, M. A., Nisula, B. C. (1983) Immunosuppressive activity of human chorionic gonadotrophin preparations in vivo: evidence for gonadal dependence Cell. Immunol. 82,334-342[CrossRef][Medline]



This article has been cited by other articles:


Home page
 J. Leukoc. Biol.Home page
H. Wan, C. G. van Helden-Meeuwsen, C. Garlanda, L. M. E. Leijten, V. Maina, N. A. Khan, H. A. Drexhage, A. Mantovani, R. Benner, and M. A. Versnel
Chorionic gonadotropin up-regulates long pentraxin 3 expression in myeloid cells
J. Leukoc. Biol., November 1, 2008; 84(5): 1346 - 1352.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0207092v1
82/4/926    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wan, H.
Right arrow Articles by Kiekens, R. C. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wan, H.
Right arrow Articles by Kiekens, R. C. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS