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Biology of Reproduction 62, 1728-1737 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Regular Article

Interferon-Tau Stimulates Granulocyte-Macrophage Colony-Stimulating Factor Gene Expression in Bovine Lymphocytes and Endometrial Stromal Cells1

Vincent Emonda,b, Éric Asselina,b, Michel A. Fortiera,b,c, Bruce D. Murphyd, and Raymond D. Lambert2,,a,b,c

a Unité de Recherche en Ontogénie et Reproduction, Centre de Recherche du Centre Hospitalier de l'Université Laval, Ste-Foy, Québec, Canada G1V 4G2 b Centre de Recherche en Biologie de la Reproduction, Université Laval, Ste-Foy, Québec, Canada G1V 4G2 c Département d'Obstétrique et Gynécologie, Université Laval, Ste-Foy, Québec, Canada G1V 4G2 d Centre de Recherche en Reproduction Animale, Université de Montréal, St-Hyacinthe, Québec, Canada J2S 7C6

ABSTRACT

Interferon-tau (IFN-{tau}), the antiluteolytic signal produced by the trophoblast prior to implantation in ruminants, exhibits immunomodulatory properties. It stimulates the production of prostaglandin (PG) E2 in bovine endometrial cells via the induction of cyclooxygenase-2 (COX-2). We previously demonstrated that preconditioning lymphocytes with PGE2 increases the expression of granulocyte–macrophage colony-stimulating factor (GM-CSF), a cytokine that promotes conceptus growth and survival. Our goal in the present study was to evaluate the impact of IFN-{tau} on the expression of GM-CSF in bovine peripheral blood lymphocytes (PBL) and endometrial epithelial and stromal cells. Changes in PGE2 production and mRNA levels of COX-2 were also studied in PBL in response to IFN-{tau}. Gene expression was estimated by semiquantitative reverse transcription–polymerase chain reaction and Northern analysis. The expression of GM-CSF in PBL was stimulated by treatment with IFN-{tau}. Furthermore, GM-CSF mRNA levels were increased after preconditioning PBL for 3 days with IFN-{tau}, followed by a 12-h restimulation without IFN-{tau}. Inhibition rather than stimulation of PGE2 production and COX-2 expression in PBL during treatment with IFN-{tau} suggests a direct effect on GM-CSF expression. Moreover, GM-CSF expression was stimulated in uterine stromal cells in response to IFN-{tau}. This study provides the first evidence for stimulation of GM-CSF expression by IFN-{tau} in both leukocytes and endometrial stromal cells. In view of the role of GM-CSF on fetal growth and survival, these results support the hypothesis that the conceptus mediates accommodation mechanisms in the uterus during early pregnancy by modulating the expression of beneficial cytokines at the fetomaternal interface.

INTRODUCTION

Under normal conditions, the conceptus (the fetus and its membranes) expresses paternal antigens but is not rejected by the maternal immune system. Nevertheless, immune-induced resorptions have been reported in mice [1–4]. Studies in mice and humans have demonstrated that immunomodulatory mechanisms are present during the periimplantation period. The conceptus escapes destruction by immune effector cells, first by expressing atypical MHC (major histocompatibility complex) molecules such as HLA-G [5, 6] that protect the invasive trophoblast against attack by cytotoxic lymphocytes (CTL) [7] and natural killer (NK) cells [8]. Second, the conceptus produces factors that prevent the activation of local cytotoxic cells, that induce changes in leukocyte phenotype [9, 10] and that stimulate production of beneficial cytokines [11, 12]. Besides inactivation of some immune functions, paracrine/autocrine messengers produced by leukocytes, uterine cells, and the conceptus induce a shift toward T-helper (Th) 2 responses and production of growth factors at the fetomaternal interface [13, 14]. Together, these events ensure not only the survival but also the growth of the conceptus. These mechanisms have particular relevance in species with hemochorial placentation, such as rodents and primates, where the trophoblast invades deep into the endometrium and is bathed in the maternal bloodstream, but they may also play a significant role in species with more superficial placentation.

Few studies have concentrated on immunomodulatory events during the peri-implantation period in ruminants [15]. These mammals display a placentation defined as syndesmochorial or synepithelial that is less invasive than its hemochorial counterpart in rodents and humans [16–18]. For this reason, the role of the maternal immune system in these species might be less determining for the success of early gestation. There is evidence, however, that local immunity plays an important role in the fate of the ruminant conceptus. Bovine embryos express paternal MHC molecules well in advance of implantation [19, 20]. Moreover, embryo transfers in which there is histoincompatibility or semicompatibility between embryo and surrogate dam have been shown to be more successful than those where MHC are identical or compatible [21]. Furthermore, immune cells believed to play key roles during pregnancy, namely large granulated lymphocytes (LGL) and {gamma}{delta} T cells, have been reported in bovine [22–24] and ovine uteri during pregnancy [23, 25, 26]. In the mouse, T cells of the {gamma}{delta} subset recognize trophoblastic cells and are activated by them [27]. It is also notable that in the sheep, LAK (lymphokine-activated killer) cells exert lytic damage upon preattachment conceptuses [28]. Therefore, immunomodulators such as TGF (transforming growth factor) ß [29] and PG (prostaglandin) E2 [9, 30, 31], which impede LAK formation and activity, may play a key role in conceptus protection.

Interferons (IFNs) of trophoblast origin were first detected in ruminants [32–36] and were later found in other species [37, 38]. In ruminants, IFN-{tau}, known as trophoblastin or trophoblast protein-1 (TP-1) until sequence and structural homologies directed it into the IFN family [32–34, 36], is believed to be the pregnancy recognition signal in these species. Interferon-tau prevents luteolysis, at least in part by inhibiting estrogen receptor expression, thus preventing estrogen stimulation of the oxytocin receptor and impeding the pulsatile release of PGF2{alpha} [39, 40]. Output of IFN-{tau} peaks just prior to the events that signal early implantation (days 15–19) [41]. As with other IFNs, IFN-{tau} inhibits cell proliferation [42–44] and exhibits antiviral activity [45]. Interestingly, IFN-{tau} is generally less cytotoxic than other IFNs [43, 46] and also differs from them in that it enhances fetal survival when administered to resorption-prone mice [47, 48], even though no murine equivalent of IFN-{tau} is known. Furthermore, IFN-{tau} stimulates cyclooxygenase (COX)-2 gene expression and PGE2 production in bovine endometrial epithelial and stromal cells in vitro [49, 50]. Because PGE2 stimulates granulocyte–macrophage colony-stimulating factor (GM-CSF) expression in peripheral blood lymphocytes (PBL) [11, 12], IFN-{tau} is believed to stimulate GM-CSF indirectly in uterine lymphocytes via the stimulation of PGE2 production in endometrial cells. Although it is known that endometrial cells synthesize GM-CSF [51], the expression of GM-CSF in bovine PBL and endometrial cells in response to IFN-{tau} has not yet been studied.

The effects of recombinant (r) ovine (o) IFN-{tau} on bovine PBL were assessed in terms of DNA synthesis, gene expression of GM-CSF and COX-2, and PGE2 production. Changes in GM-CSF steady-state mRNA levels in cultured bovine endometrial epithelial and stromal cells were also evaluated. The results suggest that, during early bovine gestation, IFN-{tau} modulates local immune functions at the fetomaternal interface by regulating the expression of GM-CSF, a cytokine known to stimulate growth of the conceptus [52–55].

MATERIALS AND METHODS

Materials

Tissue culture plates and conical tubes were purchased from Becton Dickinson (Lincoln Park, NJ) and Percoll from Pharmacia Biotech (Baie d'Urfé, Québec, Canada). Streptomycin and penicillin were obtained from ICN Biochemicals (Aurora, OH). For PBL cultures, RPMI-1640 was supplied by Gibco BRL (Burlington, Ontario, Canada) and fetal bovine serum (FBS) by Hyclone Laboratories Inc. (Logan, UT). For cultures of uterine endometrial cells, RPMI-1640 was purchased from ICN Biochemicals and FBS from Flow Laboratories Inc. (Missisauga, Ontario, Canada). Hank's balanced salt solution (HBSS), TRIZOL and reverse transcription–polymerase chain reaction (RT-PCR) enzymes and reagents were obtained from Gibco BRL. The [methyl-3H]TdR was supplied by NEN (Boston, MA), the deoxynucleotide triphosphates by Boehringer Mannheim (Laval, Québec, Canada), and QIAex and QIAquick kits by QIAgen (Chatsworth, CA). The tracer for PGE2 used in the enzyme immunoassay (EIA) was purchased from Cayman Chemical (Ann Arbor, MI) and indomethacin from Sigma (Oakville, Ontario, Canada).

Production of Recombinant Ovine IFN-{tau}

Ovine and bovine (b) IFN-{tau} are related molecules that show 80% amino acid homology [35, 36]. They have consistent biological activities when used across species [49], and luteolysis can be delayed in the cow after roIFN-{tau} injection [56]. For these reasons, roIFN-{tau} [57], kindly provided by Dr. Fuller W. Bazer (College Station, TX), was used in this study. The antiviral activity of roIFN-{tau} was 1 x 108 U/mg protein. Concentrations in this study varied from 0.01 to 10 000 ng/ml and were similar to those used in previous experiments [49, 50].

Preparation of Bovine PBL

Blood was collected from cows in a solution of citrate phosphate dextrose adenine and PBL were prepared by using a 44% Percoll:HBSS (v/v) density gradient, as described previously [12]. Equal volumes of blood and diluted Percoll were added to a 50-ml conical tube and centrifuged at 800 x g at room temperature. Cells were separated into three phases. The PBL that migrated to the middle phase, between the erythrocyte and plasma phases, were collected, washed with HBSS, and resuspended in RPMI-1640 containing streptomycin (100 ng/ml), penicillin (100 IU/ml), and decomplemented FBS (10%).

Peripheral Blood Lymphocyte Culture: Direct Treatment

The PBL were plated with 5 µg/ml concanavalin A (Con A; Laboratoire Mat Inc., Beauport, Québec, Canada), FBS (6.7% v/v), and RPMI-1640. Concentrations (0.01–1000 ng/ml) of roIFN-{tau} were added. The roIFN-{tau} vehicle (10 mM Tris/0.25 M NaCl, pH 7.5; 0.005% v/v final) had no measurable effect on any of the parameters evaluated. The plates were incubated at 37°C under a humidified atmosphere and 5% CO2, until harvested for DNA synthesis assay (48 h) or RNA extraction (6–24 h).

Peripheral Blood Lymphocyte Culture: Preconditioning

The PBL were plated with 5 µg/ml Con A (Laboratoire Mat Inc.), FBS (6.7% v/v), and RPMI-1640, in the presence (two experiments) or absence of indomethacin (1 µM). Concentrations (0.01–100 ng/ml) of roIFN-{tau} were added and cells were incubated for 72 h. The cells were collected, pooled, and centrifuged, washed with HBSS, and resuspended in RPMI containing 10% FBS. They were then redistributed with Con A (5 µg/ml), FBS (6.7% v/v), and RPMI-1640 in flatbottom plates that were reincubated at 37°C under a humidified atmosphere and 5% CO2, until harvested for DNA synthesis assay (48 h), PGE2 measurements (72 h), or RNA extraction (12 h).

Isolation and Culture of Endometrial Cells

Early cycle bovine uteri (Days 1–5), as defined by ovarian morphology [58], were collected at the slaughterhouse within 15 min of death. Endometrial epithelial and stromal cells were isolated and cultured separately in six-well plates, as described previously [59]. The purity of stromal cell cultures was increased by changing the culture medium (RPMI-1640 + 10% FBS depleted of steroids by dextran-charcoal extraction) 18 h after plating, at which time selective attachment of stromal cells had occurred. Medium was changed every 2 days, and it was observed that both epithelial and stromal cells reached confluence after 6–7 days in culture. To ensure complete differentiation, cells were cultured for a further 5 days. We have previously demonstrated that cells cultured according to this regime retain their functional properties [49, 50, 59–61]. Cells were treated by replacing the medium with 2 ml of fresh serum-free RPMI-1640 containing roIFN-{tau} (1–10 000 ng/ml) or vehicle as control, and they were incubated at 37°C under a humidified atmosphere and 5% CO2. Culture medium and cells were recovered after 24 h for PG measurements and RNA extraction.

Incorporation of [3H]TdR

Before harvesting, PBL were observed under a microscope to detect aggregation and blastogenesis, two qualitative parameters related to lymphocyte activation. The PBL (2 x 105 cells per well) were cultured for 24 h as described above, with or without preconditioning, in 96-well flatbottom plates in a final volume of 0.225 ml per well. After addition of 0.2 µCi [3H]TdR (2.0 Ci mmol/L), incubation was resumed for a further 24 h. The cells were harvested on glass fiber filters (Wallac, Turku, Finland) with a multiple cell harvester (Skatron, Lier, Norway), and the incorporated radioactivity was evaluated by liquid scintillation counting.

Enzyme Immunoassay of PGE2

Levels of PGE2 were measured in supernatants after 72 h of PBL culture without preconditioning or after 24 h of treatment of endometrial cells. An EIA technique employing acetylcholinesterase-linked PG tracers and rabbit anti-PGE2 [62] was used as described previously [61].

RNA Extraction

The PBL (2 x 106 cells per well) were cultured as described above, with or without preconditioning, in 24-well flatbottom plates in a final volume of 2.25 ml per well. The PBL were recovered and pooled after incubations of 6 h (COX-2) or 24 h (GM-CSF) for the direct treatment or after 12 h (GM-CSF) for preconditioned cells. These times were retrospectively chosen, based on preliminary experiments, for maximum expression of COX-2 (results not shown) and GM-CSF [12]. The PBL were pelleted by centrifugation at 800 x g and then lysed in TRIZOL reagent (1 ml/107 cells). Endometrial cells were directly lysed in six-well plates with 1 ml of TRIZOL per well. Cell lysates were stored at -80°C and processed within 1 mo. Total RNA was extracted by using TRIZOL according to the manufacturer's instructions. The RNA samples were resuspended in water treated with diethylpyrocarbonate (0.05% v/v) and stored at -80°C. Before analysis, RNA was quantified by measuring absorbance at 260 nm.

Reverse Transcription–Polymerase Chain Reaction Analysis

The RT-PCR was used to evaluate mRNA abundance in bovine PBL and endometrial cells according to a protocol previously described [12, 50]. Total RNA samples (400 ng) were reverse transcribed with Muloney murine leukemia virus reverse transcriptase (200 U RTase) and oligo-dT primers (0.2 µg) in a final volume of 20 µl. Reaction volumes were then brought to 65 µl. Gene expression was determined using PCR amplification of the cDNAs. The primers employed and optimization of the PCR process has previously been described [12, 50]. Each reaction was run with 4 µl of RT template or negative control and Taq DNA polymerase (1.5 U) in a final volume of 50 µl. The PCR amplifications were achieved following 26–30 cycles for GM-CSF, 38–40 cycles for COX-2, and 22–28 cycles for ß-actin. Expected PCR product lengths were 421 bp for GM-CSF, 449 bp for COX-2, and 349 bp for ß-actin, and respective identification was confirmed by restriction digestion (GM-CSF, ß-actin, COX-2) or sequencing (COX-2). The PCR products were quantified by image analysis using the AlphaImager 2000 software (Alpha Innotech Corporation, San Leandro, CA) through the linear region of the detection curve. For graphical purposes, the intensity of each band was normalized to the intensity of the corresponding ß-actin band as an internal control. A control without RTase was performed at the same time to ensure absence of contaminating DNA in the RNA templates and was negative for each amplification.

Northern Blot Analysis

Specific probes were prepared from PCR products excised from the gel and purified with QIAex using the manufacturer's protocol. The [{alpha}-32P]dCTP-labeled DNA probes produced by random priming using 100 ng DNA and the T7 Quickprime kit (Pharmacia Biotech) were purified by QIAquick following the manufacturer's procedure. Total resultant specific activity averaged 50 µCi/µg. Total RNA samples (15–20 µg) were separated by electrophoresis on a formaldehyde-containing agarose gel and transferred by capillary action to a nylon membrane (QIAgen). After prehybridization at 42°C in a 50% formamide solution, the membrane was hybridized overnight at 42°C in the same solution to which were added [{alpha}-32P]dCTP-labeled DNA probes. Membranes were washed (0.1% SDS, 0.1x standard saline citrate, 65°C) and exposed to x-ray film with an intensifying screen at -80°C for 48–96 h (GM-CSF) and 2 h (ß-actin). Autoradiograms were quantified by image analysis using the AlphaImager 2000 software through the linear region of the detection curve. For graphical display, the intensity of each band was normalized to the intensity of corresponding ß-actin band as a control. Membranes were stripped between hybridizations by treatment with a boiling 0.1% SDS solution. It should be noted that the design of the present experiments (RT-PCR and Northern analysis) did not allow determination of whether the variation in mRNA levels was due to a higher rate of transcription or to a lower rate of mRNA degradation.

Statistical Analysis

For [3H]TdR incorporation studies, each treatment was run in three replicates, and data were expressed as percentages of corresponding control without roIFN-{tau}. For gene expression studies, data were expressed as fold increases or percentages in relation to corresponding control without roIFN-{tau}. The RT-PCR analysis included three replicates per treatment, and two replicates per treatment were used for Northern analysis. Statistical analyses, including calculations of the means, SEM, and analysis of variance, were performed using super ANOVA software (Abacus Concepts, Berkeley, CA). Experiment, treatment, and interaction between experiment and treatment were included in the model as sources of variation. When the P value was less than 0.05 after ANOVA and after individual posthoc comparisons with the Student-Newman-Keuls test, treatments were considered statistically significant. Actin, the internal control of gene expression analysis, was included in the model as a covariate and was not significantly altered by treatment, and remained consistent with the expression pattern of GAPDH or 18s RNA ribosomal unit (results not shown).

RESULTS

Concanavalin A-Stimulated Bovine PBL Treated with roIFN-{tau} Exhibit Reduced Proliferation and Increased GM-CSF Gene Expression

Addition of roIFN-{tau} to PBL cultured with Con A reduced DNA synthesis in a concentration-dependent manner (Fig. 1A). A plateau in the decline was maintained in the presence of 1 ng/ml roIFN-{tau}, at 73 ± 3% of inhibition (P < 0.01 compared to control without roIFN-{tau}). Increasing amounts of roIFN-{tau} decreased cell aggregation and lowered the percentage of blastic cells (data not shown), confirming the inhibition of DNA synthesis. As shown in Figure 1B and C, levels of GM-CSF transcripts in lymphocytes were stimulated by direct treatment with roIFN-{tau} (P < 0.01 compared to control without roIFN-{tau}). A 1.5 ± 0.1-fold increase over control was obtained at 10 ng/ml, reaching 1.9 ± 0.2-fold at 100 ng/ml, and 2.1 ± 0.1-fold at 1000 ng/ml. Accordingly, at a concentration of 100 ng/ml, a 2.0 ± 0.2-fold increase of GM-CSF mRNA over control values (P < 0.05 compared to control without roIFN-{tau}) was observed using Northern analysis (Fig. 1D,E).



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  		FIG. 1. Relative inhibition of DNA synthesis and stimulation of GM-CSF gene expression in bovine Con A-stimulated PBL in response to roIFN-{tau}. Control without roIFN-{tau} is adjusted to a value of 100% or 1. **P < 0.01; *P < 0.05 compared to control. (A) Levels of [3H]TdR uptake were measured after 48 h of culture. The control value averaged 15 000 cpm. Values are means (±SEM) of n (three to seven) experiments with three replicate wells. For 100 ng/ml roIFN-{tau}, n = 7; for 1–10 ng/ml, n = 6; for 0.1 ng/ml, n = 4; and for 0.01 ng/ml, n = 3. (B–E) Bovine PBL were cultured for 24 h to measure GM-CSF gene expression. (B) The RT-PCR products of GM-CSF (28 cycles) and ß-actin (24 cycles) are shown after electrophoresis. In this and subsequent figures, pictures illustrate a single representative experiment. (C) Using image analysis, the ratios of GM-CSF:ß-actin signals were determined for all RT-PCR experiments. Values are means (±SEM) of n (two to three) experiments with three replicates. For 10–100 ng/ml, n = 3; and for 0.01–0.1–1–1000 ng/ml roIFN-{tau}, n = 2. (D) Northern blots of GM-CSF and ß-actin are shown. (E) Using image analysis, the ratios of GM-CSF:ß-actin signals were determined for all Northern experiments. Values are means (±SEM) of two experiments with two replicates

Bovine PBL Preconditioned with Con A and roIFN-{tau} Also Exhibit Reduced Proliferation and Increased GM-CSF Gene Expression After Restimulation Without roIFN-{tau}

Similar results were observed with Con A-stimulated PBL following preconditioning with roIFN-{tau} (Fig. 2). Inhibition of DNA synthesis (Fig. 2A) fell 76 ± 5% below control values (P < 0.01 compared to control without roIFN-{tau} in the preconditioning). Again, cell aggregation and percentage of blastic cells were reduced. A significant increase (P < 0.01 compared to control without roIFN-{tau} in the preconditioning) in the abundance of mRNA coding for GM-CSF (Fig. 2B,C) was reached after preconditioning PBL with 10 times less roIFN-{tau} than the dose used in the direct treatment (Fig. 1B,C). This stimulation over control values was of 1.7 ± 0.1-fold at a concentration of 1 ng/ml, reaching 2.6 ± 0.2-fold at 100 ng/ml. This was confirmed by Northern analysis (Fig. 2D,E), with an increase in GM-CSF transcripts of 3.3 ± 0.5-fold over control (P < 0.05 compared to control without roIFN-{tau} in the pretreatment).



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  		FIG. 2. Relative inhibition of DNA synthesis and stimulation of GM-CSF gene expression in bovine Con A-stimulated PBL preconditioned with roIFN-{tau} for 72 h. Control without roIFN-{tau} in the pretreatment is adjusted to a value of 100% or 1. **P < 0.01 compared to control. (A) After preconditioning with roIFN-{tau}, levels of [3H]TdR uptake were measured after another 48 h of culture with Con A alone. The control value averaged 32 000 cpm. Values are means (±SEM) of n (three to seven) experiments with three replicate wells. For 1–10–100 ng/ml roIFN-{tau}, n = 6; for 0.1 ng/ml, n = 4; and for 0.01 ng/ml, n = 3. (B–E) After preconditioning with roIFN-{tau}, bovine PBL were further cultured for 12 h with Con A alone to measure GM-CSF gene expression. (B) The RT-PCR products of GM-CSF (26 cycles) and ß-actin (22 cycles) are shown after electrophoresis. (C) Using image analysis, the ratios of GM-CSF/ß-actin signals were determined for all RT-PCR experiments. Values are means (±SEM) of n (three to six) experiments with three replicates. For 1–10–100 ng/ml roIFN-{tau}, n = 6; for 0.1 ng/ml, n = 4; and for 0.01 ng/ml, n = 3. (D) Northern blots of GM-CSF and ß-actin are shown. (E) Using image analysis, the ratios of GM-CSF/ß-actin signals were determined for all Northern experiments. Values are means (±SEM) of four experiments with two replicates

Induction of GM-CSF Gene Expression by roIFN-{tau} Does Not Result from PGE2 Production

In order to assess whether PGE2 is involved in the modulation of GM-CSF expression by bovine PBL preconditioned with 100 ng/ml roIFN-{tau}, COX-2 mRNA levels and PGE2 production were evaluated. As illustrated in Fig. 3A and B, addition of indomethacin decreased by 44 ± 5% (P < 0.01 compared to control with roIFN-{tau} without indomethacin) GM-CSF mRNA levels in PBL preconditioned with roIFN-{tau}. An equivalent reduction of 45 ± 3% was obtained upon addition of indomethacin to the preconditioning medium devoid of roIFN-{tau} (P < 0.05 compared to control without roIFN-{tau} and indomethacin). Indomethacin did not hinder [3H]TdR incorporation, suggesting that it is not cytotoxic at the concentration used (results not shown). The EIA measurements confirmed the inhibitory effect of indomethacin on PGE2 production (results not shown). The relative stimulation of GM-CSF gene expression (Fig. 3 A,B) caused by pretreatments with roIFN-{tau} in the presence or absence of indomethacin did not differ, being 2.7 ± 0.2-fold over control without indomethacin (P < 0.01), and 2.8 ± 0.2-fold over control with indomethacin (P < 0.01). Additionally, roIFN-{tau} did not significantly increase COX-2 mRNA levels (Fig. 4 A,B) or PGE2 production (Fig. 4C) in PBL. On the contrary, both COX-2 transcripts and PGE2 synthesis were decreased after treatment with IFN-{tau} (P < 0.05 compared to control without roIFN-{tau}). Both parameters plateaued in the decline at around 40% inhibition at a dose of 10 ng/ml. Thus, experiments depicted in Figs. 3 and 4 show that GM-CSF up-regulation after pretreatment with roIFN-{tau} is not induced through PGE2 stimulation during the preconditioning.



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  		FIG. 3. Inhibitory effects on GM-CSF mRNA levels caused by indomethacin in the preconditioning medium of bovine Con-A-stimulated PBL. Cells were pretreated in the presence or absence of roIFN-{tau} (100 ng/ml) or indomethacin over 72 h. The PBL were then stimulated with Con A alone during 12 h to measure GM-CSF gene expression. Values are means (±SEM) of two experiments (C indicates control without roIFN-{tau} or indomethacin, and is adjusted to a value of 1). (A) The RT-PCR products of GM-CSF (26 cycles) and ß-actin (22 cycles) are shown after electrophoresis. (B) Using image analysis, the ratios of GM-CSF:ß-actin signals were determined for all RT-PCR experiments. aP < 0.01 compared to Con A alone; bP < 0.05 compared to Con A alone; cP < 0.01 compared to Con A + indomethacin; dP < 0.01 compared to Con A + roIFN-{tau}



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  		FIG. 4. Gene expression of COX-2 and PGE2 production are not stimulated in bovine Con-A induced PBL in response to roIFN-{tau}. Control without roIFN-{tau} is adjusted to a value of 100%. **P < 0.01; *P < 0.05 compared to control. (A) Bovine PBL were cultured for 6 h to measure COX-2 mRNA levels. The RT-PCR products of COX-2 (39 cycles) and ß-actin (24 cycles) are shown after electrophoresis. (B) Using image analysis, the ratios of COX-2/ß-actin signals were determined for all RT-PCR experiments. Values are means (±SEM) of n (two to three) experiments with three replicates. For 10–100 ng/ml, n = 3; and for 0.1–1–1000 ng/ml roIFN-{tau}, n = 2. (C) Bovine PBL were cultured during 72 h to measure PGE2 synthesis. Values are means (±SEM) of n (two to three) experiments with three replicates. For 10–100 ng/ml, n = 3; and for 0.01–0.1–1 ng/ml roIFN-{tau}, n = 2

GM-CSF Gene Expression Is Stimulated in Stromal Cells of the Bovine Endometrium in Response to roIFN-{tau}

To verify whether GM-CSF expression in bovine endometrial cells [51] is regulated by roIFN-{tau}, both epithelial and stromal cell types were tested. The controls without RTase were negative for each amplification. The basal GM-CSF expression in epithelial cells (Fig. 5) was not affected by roIFN-{tau} treatment (P < 0.3). In contrast, endometrial stromal cells that express low steady-state levels of this transcript readily responded to a 5000-ng/ml roIFN-{tau} treatment (Fig. 6). The 10 000-ng/ml dose caused a 2.6 ± 0.4-fold increase over control (P < 0.05). This finding was confirmed by Northern analysis (Fig. 6 C,D), where a 2.4 ± 0.3-fold increase of GM-CSF over control values was observed in stromal cells (P < 0.01). As a control for roIFN-{tau} stimulation, PGs in the culture medium were measured [49, 50]. The IFN-{tau} induced a concentration-dependent increase of PGE2 production in epithelial cells and an increase of both PGE2 and PGF2{alpha} in stromal cells (results not shown).



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  		FIG. 5. GM-CSF gene expression is not regulated in bovine uterine epithelial cells in response to roIFN-{tau}. Epithelial cells were cultured for 24 h to measure GM-CSF mRNA levels. Control without roIFN-{tau} is adjusted to a value of 1. (A) The RT-PCR products of GM-CSF (30 cycles) and ß-actin (28 cycles) are shown after electrophoresis. (B) Using image analysis, the ratios of GM-CSF:ß-actin signals were determined for all RT-PCR experiments. Values are means (±SEM) of n (two to three) experiments. For 10, 100, 1000, 5000, and 10 000 ng/ml roIFN-{tau}, n = 3; and for 1 ng/ml, n = 2. (C) Northern blots of GM-CSF and ß-actin are shown. (D) Using image analysis, the ratios of GM-CSF:ß-actin signals were determined for all Northern experiments. Values are means (±SEM) of two experiments with two replicates



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  		FIG. 6. Gene expression of GM-CSF is up-regulated in bovine uterine stromal cells in response to roIFN-{tau}. Stromal cells were cultured for 24 h to measure GM-CSF mRNA levels. Control without roIFN-{tau} is adjusted to a value of 1. **P < 0.01; *P < 0.05 compared to control. (A) The RT-PCR products of GM-CSF (30 cycles) and ß-actin (28 cycles) are shown after electrophoresis. (B) Using image analysis, the ratios of GM-CSF:ß-actin signals were determined for all RT-PCR experiments. Values are means (±SEM) of n (two to four) experiments with two replicates. For 100 ng/ml roIFN-{tau}, n = 4; for 1, 1000, and 5000 ng/ml, n = 3; and for 10 and 10 000 ng/ml, n = 2. (C) Northern blots of GM-CSF and ß-actin are shown. (D) Using image analysis, the ratios of GM-CSF:ß-actin signals were determined for all Northern experiments. Values are means (±SEM) of three experiments with two replicates

DISCUSSION

The present study is the first demonstration that IFN-{tau} regulates GM-CSF expression in the bovine immune and reproductive systems. The IFN-{tau} specifically originates from the conceptus and is produced over a limited period during early gestation [41]. This study and our previous data [12] suggest that, in ruminants, the conceptus has the capacity for local modulation of the production of cytokines and growth factors that, in turn, may sustain development and maintain pregnancy.

In this investigation, direct in vitro treatments with roIFN-{tau} mimicked acute exposure of immune and uterine cells to peak levels of IFN-{tau} produced in vivo between days 15 and 19 [41]. The PBL responded to this treatment with reduced proliferation, as previously reported [42, 44], and with concurrent stimulation of GM-CSF mRNA levels. The high basal GM-CSF expression in endometrial epithelial cells was not regulated by roIFN-{tau}, however. In contrast, GM-CSF gene expression was readily stimulated in endometrial stromal cells. It is interesting to note that leukocytes are about a thousand times more sensitive to IFN-{tau} than stromal cells. Overall, this suggests that before implantation, the embryo is provided with an important growth factor produced by epithelial cells, and later, resident leukocytes and stromal cells quickly respond to IFN-{tau} by a further supply of GM-CSF.

Experiments with preincubation of PBL with roIFN-{tau}, followed by a restimulation with Con A, were used to simulate the conditions that intraendometrial immune cells are subjected to after the major peak of IFN-{tau}, which lasts around 3–4 days. We show that, even after removal of roIFN-{tau} from the culture medium, up-regulation of GM-CSF transcripts and reduced proliferation are maintained. This suggests that preconditioned PBL differentiated in response to IFN, as described in other systems [63, 64]. Pretreatments with roIFN-{tau} but without Con A, followed by the same restimulation with Con A, were also performed (results not shown); however, only PBL pretreated with both IFN-{tau} and Con A showed stimulation of GM-CSF expression. This suggests that lymphocytes must be activated in order to respond to IFN-{tau} by an increase in GM-CSF. Such a stimulation is likely to occur in vivo, as {gamma}{delta} T cells are activated during pregnancy in sheep [26], and they recognize trophoblast cells and respond to them in mice [27]. By reducing proliferation and nonetheless activating bovine PBL toward increased GM-CSF expression, it appears that roIFN-{tau} induces functional differentiation-dependent changes in these cells. Indeed, while GM-CSF production by human PBL is unaffected by acute treatment with either TGFß2 and PGE2, it is dramatically increased after a 72-h preconditioning with the same factors [11]. In human cells, the rise in GM-CSF is preceded by down-regulation of TCR, CD4 [10], and CD8 [65], supporting the hypothesis that preconditioning PBL with immunomodulators brings about differentiation and consequent alteration of secretory functions. The cell lineages of the leukocytes involved in GM-CSF production during bovine gestation remain to be identified. LGL might be good candidates because they constitute an important source of GM-CSF in humans [66] and because their presence has been demonstrated in the epithelial layer of the bovine endometrium [22–24].

Bovine uterine epithelial and stromal cells respond to rIFN-{tau} by increasing COX-2 mRNA levels and PGE2 production [49, 50]. Because PGE2 stimulates GM-CSF expression in bovine PBL [12], it was possible that the effects of roIFN-{tau} on GM-CSF expression in bovine PBL were indirectly mediated. Indeed, bovine PBL respond to either IFN-{tau} or PGE2 by reduction in cell proliferation and stimulation of GM-CSF expression [12]. The present study demonstrates that roIFN-{tau} increases neither COX-2 mRNA levels nor PGE2 production in bovine PBL, arguing for a direct effect of roIFN-{tau} to increase GM-CSF mRNA levels in these cells. On the other hand, GM-CSF gene expression is reduced following pretreatment with indomethacin, suggesting an autocrine action of PGE2 in maintaining the basal production of GM-CSF in bovine PBL. Our data thus suggest that regulation of GM-CSF by PGE2 and IFN-{tau} are independent and additive. Indeed, the relative stimulation of GM-CSF after IFN-{tau} preconditioning is identical with or without indomethacin. Also, the relative inhibition of GM-CSF after indomethacin preconditioning is identical in the presence or absence of roIFN-{tau}. Together, these findings demonstrate that PGE2 is not involved as an intermediary messenger in PBL response in this system.

The growth factor GM-CSF plays a crucial role in successful pregnancy. The positive impact of this cytokine/growth factor on murine fertility has been demonstrated by the null gene mutation [67]; GM-CSF promotes conceptus growth and development both in vivo and in vitro [52–54]. In particular, in vitro development of early bovine embryos is enhanced [55] and ovine Day 14/16 conceptus may respond to GM-CSF [68–70]. Moreover, GM-CSF has been detected in uterine flushings of the pregnant cow [51], and its presence in the uterine lumen may create a milieu that is beneficial for the development of the conceptus.

The relative contribution of cytokines from immune versus nonimmune cells of the uterus to conceptus growth remains speculative. The immunohistochemical demonstration of GM-CSF in bovine uterine epithelial cells during the estrous cycle [51] concurs with the high basal gene expression of GM-CSF in these cells in vitro. Isolated cells in the stroma of the bovine uterus, staining for GM-CSF, have been associated with leukocytes [51]. This fits with the present demonstration of the expression of GM-CSF by bovine PBL. However, the physiological relevance of uterine immune cells as essential components of successful gestation is still unclear [71]. In this context, regulation of GM-CSF expression in uterine stromal cells, indicates that the contribution of nonimmune compartments to the production of placentoembryotrophic factors, relative to that of immune cells, might be higher than previously expected. Under normal conditions during early pregnancy, immune cells may mediate the communication between the conceptus and endometrial cells, permitting optimization of accommodation mechanisms. The high sensitivity of immune cells demonstrated in the present report argues for this hypothesis.

A schema to illustrate these complex interactions is presented in Fig. 7. Basal secretion of GM-CSF by uterine epithelial cells [51, 68] promotes early embryonic development [55] upon entry in the uterus. Later, IFN-{tau} production by the trophoblast (peak at Days 15–19 [41]) may be stimulated by GM-CSF from the epithelium [68–70]. Stromal cells and uterine leukocytes respond to IFN-{tau} by increasing their GM-CSF secretion, and this could result in an amplification loop. Synthesis of PGE2 is enhanced in epithelial and stromal cells in response to IFN-{tau} [49, 50] and in macrophages in response to GM-CSF [72]. The proliferation of resident leukocytes is reduced by exposure to massive amounts of IFN-{tau} and PGE2. After the decline of IFN-{tau} that occurs when trophoblastic cells begin to fuse with epithelial cells [73], GM-CSF production by maternal immune cells is maintained and increased further. Taken together, these findings suggest that the bovine conceptus orchestrates accommodation mechanisms by directly targeting maternal immune and nonimmune cells to induce beneficial cytokine production.



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  		FIG. 7. Cross-talk between the conceptus, endometrial cells, and maternal immune cells during early gestation in the cow. The IFN-{tau} is produced by the embryo and its synthesis may be stimulated by GM-CSF from epithelial cells [68–70]. The IFN-{tau} stimulates GM-CSF synthesis by uterine leukocytes and stromal cells. An amplification loop may be established between IFN-{tau} and GM-CSF; IFN-{tau} also induces an increase in PGE2 production in epithelial and stromal cells [49, 50], as well as functional changes in uterine leukocytes in parallel to those caused by PGE2 [12]. The decline in IFN-{tau} secretion is followed by increased GM-CSF production in uterine leukocytes. Overall GM-CSF stimulation probably results in conceptus growth and reorientation of the cytokine network at the fetomaternal interface.

ACKNOWLEDGMENTS

The authors thank Dr. Fuller W. Bazer for generously donating roIFN-{tau}, Dr. Louis Picard for kindly providing bovine blood, Dr. Sylvie St-Jacques for her intellectual contribution, Ms. Christine Légaré for her technical advice, and Ms. Virginie Guilbert for PGE2 measurements.

FOOTNOTES

First decision: 16 July 1999.

1 This work was supported by grant 98-ER-2421 from the Fonds pour la Formation de Chercheurs et l'Aide à la Recherche (FCAR) and grants to M.A.F. and R.D.L. each from the Natural Sciences and Engineering Research Council of Canada (NSERC). Back

2 Correspondence: Raymond D. Lambert, Ontogénie et Reproduction T1-49, Centre de Recherche du Centre Hospitalier de l'Université Laval, 2705 Blvd. Laurier, Ste-Foy, PQ, Canada G1V 4G2. FAX: 418 654 2765; ray.lambert{at}crchul.ulaval.ca Back

Accepted: January 12, 2000.

Received: June 4, 1999.

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