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research-article |
Department of Human Pathology and Oncology,3 University of Siena, 53100 Siena, Italy
Department of Obstetrics, Gynecology and Reproductive Sciences,4 Yale University School of Medicine, New Haven, Connecticut 06520
Department of Molecular and Cellular Biology,5 Harvard University, Cambridge, Massachusetts 02138
ABSTRACT
Macrophages are a major component of the leukocyte population of human pregnant endometrium. Although several crucial functions have been ascribed to these cells, the mechanisms underlying macrophage trafficking in the placental bed are poorly understood. The aim of this study was to evaluate the in vivo expression of two potentially antagonistic macrophage-targeting chemokines, colony stimulating factor 1 (CSF1, also known as M-CSF) and macrophage migration inhibitory factor (MIF), in term decidua, and to examine the effects of the inflammatory cytokines tumor necrosis factor (TNF, also known as TNF alpha) and interleukin 1beta (IL1B) on CSF1 and MIF expression in cultured decidual cells. The expression of CSF1 and MIF in term decidua was evaluated by immunohistochemistry. Cultured decidual cells were primed with estradiol (E2) or with E2 + medroxyprogesterone acetate (MPA), and then incubated with corresponding steroid(s) with or without TNF or IL1B. The levels of CSF1 and MIF protein and mRNA were assessed by ELISA and quantitative RT-PCR, respectively. Immunostaining for CSF1 and MIF was observed in term decidua. The levels of secreted CSF1 and MIF were similarly unchanged whether the decidual cells were incubated with E2 or with E2 + MPA. The CSF1 levels significantly increased in cultures exposed to E2 or E2 + MPA plus TNF or IL1B. In contrast, the MIF levels in TNF- and IL1B-treated cells were not changed significantly from the control cultures. The ELISA data were confirmed by quantitative RT-PCR analysis. These results indicate that CSF1 and MIF are involved in regulating macrophage trafficking at the fetal-maternal interface, and suggest a mechanism by which inflammatory cytokines influence pregnancy by regulating decidual macrophage infiltration.
cytokines, deciduas, immunology, pregnancy
The early pregnant human endometrium (decidua) contains a diverse population of leukocytes that mediate innate immunity, including uterine natural killer (uNK) cells (70–75%) and macrophages (20–25%). In contrast, T and B lymphocytes, which mediate adaptive immunity, account for 10% and between 1% and 3 % of the white cell population, respectively [1, 2]. In recent years, research has focused on the role played by uNK in placentation, with far less attention being directed towards decidual macrophages. Nevertheless, several crucial pregnancy-related functions have been ascribed to these leukocytes, ranging from the utero-placental response to infection and the production of cytokines, to the establishment of maternal tolerance and tissue remodeling during implantation and trophoblast invasion [3]. An excess of decidual macrophages has also been implicated in the impaired endovascular trophoblast invasion that underlies pre-eclampsia and intrauterine growth restriction [4, 5].
Histological studies have revealed that the decidual leukocyte cell population undergoes dynamic changes throughout gestation. At the implantation site, uNK cells increase in number during the first trimester, and then gradually decline until they are virtually absent at term [1]. The numbers of T and B lymphocytes increase in the decidua throughout gestation [6, 7]. In contrast, decidual macrophage numbers remain relatively invariant throughout normal gestation [1].
The mechanisms for controlling leukocyte trafficking in the placental bed are poorly understood. However, previous studies have shown that multiple chemotactic and chemostatic factors are produced at the fetal-maternal interface, supporting the concept that an elaborate chemokine network controls leukocyte recruitment and maintenance throughout normal and pathological pregnancies [8].
Colony stimulating factor 1 (CSF1; also known as macrophage-colony stimulating factor or M-CSF) is a 45–90-kDa homodimeric glycoprotein that regulates the proliferation and differentiation of the mononuclear phagocytic cell lineage. CSF1 acts via a high affinity cell surface receptor product of the CSF1R proto-oncogene to affect macrophage viability, differentiation, chemoattraction, motility, and adhesion [9]. Furthermore, CSF1 helps to maintain pregnancy by mediating trophoblast-endometrial interactions [10]. Confirmation of the crucial role played by CSF1 in pregnancy has come from studies of osteopetrotic (op/op) mice, which carry an inactivating mutation in the coding region of CSF1. These mice are depleted of circulating monocytes and tissue macrophages in several organs, including the uterus. They experience severely reduced fertility, lower implantation rates, and greater embryonic wastage compared with wild-type females [11]. Paradoxically, excess decidual macrophage infiltration has been linked to pre-eclampsia and intrauterine growth restriction in humans [4].
Macrophage migration inhibitory factor (MIF) is a 12.5-kDa cytokine that inhibits the migration and chemotaxis of macrophages [12, 13]. High steady-state levels of MIF mRNA and protein have been detected in human reproductive tissues. MIF expression has been reported in the follicular fluid and granulosa cells of the human ovary [14]. The presence of MIF in embryonic and maternal tissues has been documented in previous studies conducted by our group. In first trimester human placentas, we have detected MIF in the cytotrophoblasts of both the inner layer of the villi and in the trophoblastic cell islands [15]. More recently, we have demonstrated the presence of MIF in the glandular and stromal compartments of cycling endometrium, as well as in first trimester decidua [16]. In keeping with its known immunomodulatory functions, we have proposed the involvement of MIF in regulating macrophage accumulation in the pregnant endometrium [16].
The current study evaluated the involvement of CSF1 and MIF in the recruitment and maintenance of macrophages in human decidua. After demonstrating the presence of these two potentially antagonistic chemokines by immunohistochemical staining of sections of term decidua, we sought to elucidate the mechanisms underlying decidual CSF1 and MIF expression. To accomplish this goal, we evaluated the effects of tumor necrosis factor (TNF) and interleukin 1beta (IL1B), which are known regulators of CSF1 and MIF expression that are present at the fetal-maternal interface, on CSF1 and MIF mRNA and protein levels in monolayers of leukocyte-free decidual cells. The interactions of progestin with these inflammatory cytokines on CSF1 and MIF expression were also assessed in these cultured cells. The results suggest that CSF1 and MIF are involved in regulating macrophage trafficking in human decidua, and describe a novel mechanism through which TNF and IL1B can influence pregnancy.
Anti-human MIF and CSF1 goat polyclonal antibodies were purchased from R&D Systems (Abingdon, UK) and Santa Cruz Biotechnology (Santa Cruz, CA), respectively. Horseradish peroxidase-conjugated rabbit anti-goat antibody was obtained from Calbiochem (San Diego, CA). All the chemicals were of analytical grade (Sigma Chemical Co., St. Louis, MO).
Placentas (n = 6) were obtained following cesarean delivery due to term breech presentation. None of the patients were in labor. Approval for this study was granted by the Human Institutional Investigation Committee (HIC) of the University of Siena. Informed consent was obtained from all women. Full-thickness placental blocks were fixed in 10% buffered neutral formalin and embedded in paraffin for histology and immunohistochemistry. For each tissue, a small portion was embedded in OCT (Bio-Optica, Milan, Italy) and frozen in liquid nitrogen. Slides of each specimen were stained with hematoxylin-eosin and examined histologically by a pathologist. Only tissues from uncomplicated pregnancies were included in this study. For each specimen, a block that best represented the maternal decidua was selected for immunohistochemistry.
For MIF staining, sections (4-µm thickness) of paraffin-embedded placenta tissues were cut, deparaffinized, rehydrated, and washed in Tris-buffered saline (TBS; 20 mM Tris-HCl [pH 7.6], 150 mM NaCl). TBS was used for all subsequent washes and for dilution of the antibody. Antigen retrieval was carried out by incubating sections in 10 mM sodium citrate buffer (pH 6.0) in a microwave oven at 750 W for 5 min. For CSF1 immunostaining, frozen tissues were cut (6-µm thickness) and fixed in ice-cold acetone. Sections were rinsed in 3% hydrogen peroxide, to block endogenous peroxidase, and incubated overnight with polyclonal antibodies against MIF and CSF1 at dilutions of 1:300 and 1:50, respectively. The slides were then washed three times with TBS for 5 min, and incubated with peroxidase-labeled rabbit anti-goat antibody (Calbiochem) at a dilution of 1:2000 for 30 min. After washing three times for 5 min in TBS, the sections were incubated in diaminobenzidine tetrahydrochloride (DAKO, Copenhagen, Denmark) in TBS with 0.01% hydrogen peroxide for 15 min. The reaction was stopped by washing the sections in distilled water, and the slides were mounted and observed under a light microscope. The staining specificity for each cytokine was tested by substituting non-immune serum for the primary antibody.
Isolation and Culture of Decidual Cells
After receiving written informed consent, placentas and attached fetal membranes were obtained from patients with uncomplicated pregnancies who were undergoing repeat cesarean deliveries at term at Yale-New Haven Hospital under HIC approval. None of these patients was in labor. A small portion of each specimen was formalin-fixed, paraffin-embedded, and then examined histologically to rule out underlying inflammation. The decidua was scraped from the maternal surface of the chorion, minced, and digested in Ham F-10 plus 10% charcoal-stripped calf serum (SCS) (Flow Laboratories, Rockville, MD) that contained 25 mg/ml collagenase (200 U/mg) (Worthington Biochemical Corp., Freehold, NJ) in a shaking water bath at 37°C for 30 min. After adding 6.25 U DNase (Sigma) per ml of digestate, the incubation was continued for an additional 45 min. The final digestate was passed through a 23G needle to dissociate the remaining cell clusters. The isolated cells were centrifuged at 1500 rpm for 5 min at 4°C, and washed in Ham F-10 medium. This procedure was repeated three times and the final cell pellet was resuspended (1 g of tissue/ml) in 20% Percoll (Sigma), layered on a 60%:50%:40% discontinuous Percoll gradient, and then centrifuged at 22 000 rpm for 20 min at 4°C. The top cell layer was collected, washed, resuspended in Ham F-10, and centrifuged at 1500 rpm for 5 min at 4°C. After repeating this procedure, the resulting cell pellet was resuspended in 40% Percoll (1 g of tissue/ml), layered on a 55%:50%:40% discontinuous Percoll gradient, and centrifuged at 22 000 rpm for 20 min at 4°C. The top cell layer was washed twice in serum-free Ham F-10, and then centrifuged at 500 rpm for 5 min at 4°C. The cell pellet was resuspended in Ham F-10 plus 10% SCS and the decidual cells were counted in a hemocytometer. Trypan blue exclusion identified more than 95% of isolated cells as being viable.
Isolated decidual cells (5 x 105 cells/ml) were suspended in Basal Medium (BM), which is a phenol red-free 1:1 (v/v) mix of DMEM (Invitrogen, Carlsbad, CA) and Ham F-12 (Flow Laboratories) with 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml fungizone, and supplemented with 10% SCS. The decidual cells were seeded onto polystyrene tissue culture dishes that were precoated with 2% type B gelatin (Sigma Chemical Co.). The cultures were grown to confluence in 5% CO2 in air at 37°C, and passaged three times. Fluorescent antibody cell sorting for the presence of CD45+ demonstrated that unpassaged cultures contained 12–15% CD45+ cells, while the passaged cultures were >99% negative for this common leukocyte marker. The latter cell populations were used for the subsequent experimental cell incubations. The cultured cells were vimentin-positive and cytokeratin-negative. The cultured cells also showed decidualization-related morphological changes and expressed elevated levels of prolactin, tissue factor, and plasminogen activator inhibitor type 1 under the influence of estradiol (E2) plus medroxyprogesterone acetate (MPA).
Confluent decidual cultures, each obtained from an individual specimen, were primed for 7 days in BM supplemented with SCS that contained either vehicle control (0.1% ethanol), 10–8 M E2, 10–7 M MPA (Sigma) or E2 plus 10–7 M MPA with one change of medium. Since the circulating levels of E2 and progesterone are high during the third trimester, E2 was employed with MPA to mimic the gestational steroidal milieu. MPA was used instead of native progesterone, which is rapidly metabolized in vitro [17]. The cultures were washed twice with PBS, to remove residual serum components, and switched to a serum-free defined medium (DM) that consisted of BM plus ITS+ premix (BD Biosciences, Bedford, MA), 5 µm FeSO4, 50 µm ZnSO4, 1 nm CuSO4, 20 nm Na2SeO3, trace elements (Invitrogen), 50 µg/ml ascorbic acid (Sigma), and 50 ng/ml epidermal growth factor (BD Biosciences). The corresponding vehicle or steroid(s) with or without TNF and IL1B (R&D Systems) was added to DM.
After 24 h of incubation, the concentrations of immunoreactive CSF1 and MIF in conditioned DM were measured by specific ELISAs according to the instructions provided by the manufacturer (R&D Systems). The levels of CSF1 and MIF were normalized to the total cell culture protein content, as measured by Lowry protein assay (Bio-Rad Laboratories, Hercules, CA). The intra- and inter-assay coefficients of variation and assay sensitivity were 2.3%, 6.5% and 15.62 pg/ml and 5.0%, 6.9%, and 31.25 pg/ml, respectively, for CSF1 and MIF.
Total RNA was extracted from cultured cells with Tri Reagent (Sigma). Reverse transcription was carried out with an AMV reverse transcriptase kit (Invitrogen) on an Eppendorf Mastercycler (Eppendorf, Westbury, NY). To perform quantitative real-time RT-PCR, a standard curve was created for 500 pg to 250 ng cDNA using the Roche Light Cycler (Roche, Indianapolis, IN), with monitoring of the increasing fluorescence of the PCR products during amplification. Upon establishing the standard curve, quantitation of the unknowns was determined with the Roche Light Cycler and adjusted to the quantitative expression of the beta-actin gene (ACTB) from the corresponding unknowns. Melting curve analysis determined the specificity of the amplified products and the absence of primer-dimer formation. All of the products obtained yielded the correct melting temperatures. The following primers were synthesized and gel-purified at the Yale DNA Synthesis Laboratory, Critical Technologies: CSF1 sense primer 5'-GCTCAGCCAGATGCAA-3' and antisense primer 5'-GTCCAGGTGGTCATG-3'; and MIF sense primer 5'-GTAGCCACATGATTGG-3' and antisense primer 5'-GTTATCTCTGAAGCGC-3'. The ACTB sense and antisense primers were 5'-CGTACCACTGGCATCGTGAT-3' and 5'-GTGTTGGCGTACAGGTCTTTG-3', respectively. The expected sizes of the fragments amplified for CSF1, MIF and ACTB were 207 bp, 191 bp, and 459 bp, respectively.
Comparisons of the control and the various treatment groups were performed using the Kruskal-Wallis ANOVA on Ranks test followed by the Student-Newman-Keuls post hoc test with P < 0.05 representing statistical significance.
CSF1 and MIF Expression in Term Decidua
The distributions of the CSF1 and MIF proteins in term decidua were analyzed by immunohistochemistry. Strong immunostaining for CSF1 (Fig. 1, A and B) and MIF (Fig. 1, C and D) were observed in the decidua. Observations at higher magnification revealed that both proteins were primarily distributed in the cytoplasm. Intense immunostaining was also detected in the syncytiotrophoblast (CSF1) and cytotrophoblast (MIF) of the chorionic villi. In addition, MIF immunoreactivity was consistently found in extravillous trophoblasts (data not shown). Staining for both cytokines was eliminated by substituting non-immune serum for the primary antibodies (data not shown).
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Regulation of CSF1 and MIF Expression in Decidual Cells
As circulating levels of both E2 and progesterone are high during the third trimester, E2 was used as the control for evaluating the effects of the synthetic progestin MPA. Figure 2 indicates that in cultures maintained with E2 alone, 1 ng/ml of TNF or IL1B increased CSF1 output from 0.1 ± 0.02 ng/ml/mg protein in the control cultures to 0.54 ± 0.18 (mean ± SEM; P < 0.05) and 0.7 ± 0.14 (P < 0.05) ng/ml/mg protein, respectively. In E2 + MPA-treated cultures, CSF1 output was increased from the basal level of 0.2 ± 0.05 ng/ml/mg protein to 0.7 ± 0.14 (P < 0.05) and 0.79 ± 0.13 (P < 0.05) ng/ml/mg protein by TNF and IL1B, respectively. In contrast to the marked elevation of CSF1 elicited by these cytokines, the addition of MPA plus E2 did not significantly alter the levels of CSF1 compared to cultures treated with E2 alone, and did not alter the responses to the cytokines.
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In contrast to the effects of TNF (1 ng/ml) and IL1B on CSF1 expression, neither cytokine influenced the MIF levels in decidual cell-conditioned medium (Fig. 3). Specifically, the levels of MIF in TNF (1 ng/ml)- and IL1B (1 ng/ml)-treated cells were 15.8 ± 4.4 ng/ml/mg cell protein and 14.6 ± 3.45 ng/ml/mg cell protein, respectively, which were not significantly different than the levels in control cultures incubated with E2 alone (16.4 ± 3.2 ng/ml/mg cell protein). Similarly, in E2 + MPA-treated cultures, the basal level of MIF was 18.4 ± 4.4 ng/ml/mg protein, and this was not changed by treatment with either TNF (12.58 ± 2.0 ng/ml/mg protein) or IL1B (15.5 ± 2.4 ng/ml/mg protein). Corresponding results were obtained when: 1) the incubation period with TNF and IL1B was extended to 48 h; 2) the cytokine concentration was increased to 10 ng/ml; and 3) cultures were not treated with steroids.
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To determine whether this differential response to inflammatory cytokines was present throughout gestation, TNF and IL1B were tested on cultured first trimester decidual cells. Consistent with our observations for term decidual cells, in first trimester decidual cell cultures maintained in E2 or E2 + MPA, 1 ng/ml of TNF or IL1B increased the CSF1 output (Fig. 4), whereas these cytokines did not significantly alter the MIF protein levels (Fig. 5).
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Given the absence of a steroid effect, further evaluations of the effects of TNF and IL1B on CSF1 expression were carried out with term decidual cells that were primed with E2 + MPA. The dose-response relationships of CSF1 to each cytokine added at concentrations between 0.1 and 10 ng/ml are shown in Figure 6. Both TNF (A) and IL1B (B) elevated CSF1 output over the entire range of concentrations tested, with maximum effects for both cytokines obtained at 10 ng/ml.
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Following experimental incubations of the leukocyte-free term decidual cells, the levels of CSF1 and MIF mRNA were determined in extracted RNA samples by quantitative RT-PCR. As indicated in Figures 7 and 8, changes in the patterns of CSF1 and MIF mRNAs corresponded to those established for their respective proteins. Thus, TNF and IL1B each induced a statistically significant increase in CSF1 mRNA levels, irrespective of whether they were added with E2 or with E2 + MPA, whereas in the absence of cytokines, similar CSF1 mRNA levels were observed in parallel incubations with E2 or with E2 + MPA (Fig. 7). In contrast, the MIF mRNA levels were not significantly altered by TNF, IL1B or MPA (Fig. 8).
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The present study immunolocalized CSF1 and MIF to decidual cells in sections of term decidua, and established that leukocyte-free, cultured decidual cells from first trimester and term placentas synthesize and secrete both chemokines. These findings extend previous observations of CSF1 and MIF expression in early decidua [16, 18] to include term decidua, and suggest that these chemokines are synthesized and secreted by decidual cells throughout pregnancy. Several lines of evidence prompted us to evaluate the effects of TNF and IL1B on term decidual cells. TNF and IL1B are expressed at the fetal-maternal interface. The mRNA for TNF is present in syncytiotrophoblasts of the first trimester and term placentas and in the decidua at term [8]. Immunoreactive IL1B has been detected in villous and extravillous trophoblasts, whereas TNF and IL1B secretion has been demonstrated in explants of term placentas and decidua [8, 19].
The current results indicate that TNF and IL1B induce CSF1 output by cultured first trimester and term decidual cells. This finding suggests the existence of a novel autocrine/paracrine mechanism, through which TNF and IL1B enhance the production of the macrophage-targeting chemokine CSF1 in decidual cells throughout pregnancy in vivo. Although two recent reports have demonstrated that exposure to TNF and IL1B increases MIF protein and mRNA levels in stromal cells derived from cycling human endometrium [20, 21], the current results indicate that while MIF is synthesized and secreted at high concentrations by term decidual cells, its expression is not regulated by TNF or IL1B. Previous studies have demonstrated that MIF can induce, either directly or indirectly, the production of several proinflammatory molecules, including cytokines, nitric oxide, and prostaglandins [12]. The constitutive expression of MIF by decidual cells revealed in the present study is likely to modulate the delicate equilibrium between proinflammatory and anti-inflammatory factors, which is crucial for the maintenance of pregnancy. Thus, the absence of regulation of MIF production by decidual cells following TNF and IL1B exposure demonstrated in the current study could be an important previously undisclosed mechanism for preventing an exaggerated inflammatory response at the implantation site.
Decidual macrophages regulate trophoblast migration and proliferation, and are likely to play important roles in controlling the local maternal immune response as well as immunological rejection of the semi-allogenic embryo [3, 22]. Decidual macrophages bind to and phagocytose bacteria [23], regulate placental apoptosis, and are involved in clearance of placental apoptotic cells [3]. Conversely, macrophages are also a source of factors that may lead to adverse effects on the conceptus. Tissue macrophages synthesize and secrete the Th1 cytokines interferon-gamma and TNF, as well as the effector molecules nitric oxide and prostaglandins [24]. Consequently, mechanisms that ensure the regulation of macrophage numbers at the fetal-maternal interface are likely to play crucial roles in the establishment and maintenance of pregnancy.
CSF1 and MIF are known to exert opposite effects on macrophage chemotaxis, with CSF1 promoting and MIF inhibiting migration. Moreover, MIF specifically inhibits MCP-1-driven monocyte and macrophage migration [13]. The results of the current study support the involvement of these antagonistic cytokines in controlling the decidual macrophage population. They suggest that variability in the magnitude of CSF1 release from TNF- and IL1B-stimulated decidual cells can have physiologic or pathologic consequences. Moreover, excessive CSF1-driven accumulation of decidual macrophages at the fetal-maternal interface can be prevented by the high constitutive decidual MIF levels, resulting in tight control of the decidual macrophage population that could have an impact on fertility and pregnancy maintenance.
Previous studies have shown that pre-eclampsia is associated with augmented expression of TNF and IL1B [25, 26] and excess decidual macrophage infiltration [4]. CSF1 levels are elevated in the blood of patients prior to the onset of pre-eclamptic signs or symptoms [27]. Furthermore, increased CSF1 expression has been reported in placental tissue obtained from pre-eclamptic patients [28]. Consequently, the induction of CSF1 by TNF and IL1B during the period of endovascular trophoblast invasion may promote the infiltration of macrophages. The presence of the latter in the placental bed may be a source of TNF, which induces apoptosis in extravillous trophoblasts and restricts their invasion [5]. The resulting shallow placentation is a hallmark of pre-eclampsia. Similarly, we have reported that TNF and IL1B also induce the expression of another macrophage chemoattractant, monocyte chemoattractant protein-1, in cultured DCs [4]. More recently, we have shown that IL1B induces an array of chemokines that promote monocyte/macrophage chemotaxis [29]. Thus, TNF and IL1B may induce multiple macrophage-monocyte/macrophage-recruiting chemokines that overcome the inhibitory effects of constitutively expressed MIF.
The effects of TNF and IL1B on CSF1 levels shown in the current study delineate a novel mechanism by which inflammatory cytokines can affect pregnancy. Several lines of evidence indicate that CSF1 plays important roles in regulating placental function and growth. In the mouse, CSF1 induces placental DNA synthesis in vitro [30]. In humans, both CSF1 and the CSF1 receptor are highly expressed in the placenta [18], whereas CSF1 stimulates human trophoblast hCG production in vitro [31]. A role for CSF1 in placental and fetal growth is further supported by the observation that CSF1 levels in the amniotic fluid are decreased in patients with intrauterine growth retardation [32]. Therefore during pregnancy, IL1B and TNF may indirectly regulate placental function and growth through the induction of decidual CSF1 production.
FOOTNOTES
1This work was supported by grants from the National Institutes of Health 2 R01HD 33937-04 (C.J.L.) and 1 R01 HL070004-03 (C.J.L.), from the University of Siena (M.C. and F.A.), and from the Italian Ministry of Education and Scientific Research (P.T.). 
Correspondence: 2Frederick Schatz, Department of Obstetrics, Gynecology and Reproductive Sciences, Yale University School of Medicine, 333 Cedar Street, Room 335 FMB P.O. Box 208063 New Haven, CT. FAX: 203 785 4713; e-mail: Frederick.Schatz{at}yale.edu
Received: 26 May 2006.
First decision: 29 June 2006.
Accepted: 14 November 2006.
REFERENCES
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