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Articles |
Contributes to the Normalcy of Murine Pregnancy1
a Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada N1G 2W1
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, is a major function of most NK cell subsets. In this study, uNK cells were assessed for IFN- production. Local concentrations of IFN- were measured in the mesometrial regions of murine implantation sites between Days 6 and 16 of gestation. IFN- was detected by ELISA at all days studied in a random-bred (CD1) and an inbred (BALB/c) strain of immune-competent mouse and in two immune-deficient strains, SCID (NK+, T-, B-) and tg
26 (NK-, T-, B+). Concentrations of IFN- per implantation site peaked at Day 10 of gestation in NK+ strains but were low and relatively constant in NK- mice. To evaluate the functions of IFN- at murine implantation sites, pregnancy was studied in homozygously mated IFN--/- and IFN-R
-/- mice and their congenic controls. Primiparous but not multiparous IFN--/- mice experienced significant fetal loss. Primiparous IFN-R-/- carried full litters to term. Implantation site pathology was demonstrated in both strains of gene-deleted mice by light microscopy and ultrastructurally. This included elevated numbers of uNK cells that contained fewer and smaller granules and, after Day 10 of gestation, progressive necrosis and loss of decidua. The presence of a fetus able to produce IFN- did not modify the phenotype of pregnant IFN--/- mice. This study indicates that during murine pregnancy, uNK cells are the main source of IFN- on the mesometrial side of the uterus and that IFN- contributes to normal health of the midgestational decidua. Furthermore, evidence is presented that IFN--producing cells exist in mesometrial regions of implantation sites that are neither NK nor T cells.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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26 mice by bone marrow from SCID (NK+, T-, B-) donors confirmed their NK lineage [6] and led to their renaming as uterine natural killer (uNK) cells.
Absence of uNK cells in NK and T cell-deficient mice (tg26, IL-2Rß-/- x P56lck-/-, common cytokine chain (c)-/- and Rag-2-/- x c-/-) is associated with light-microscopic anomalies from Day 10 of gestation. These include vasculopathy within the decidua, decidual edema and acellularity, and small placental size ([8]; unpublished results). These data suggest that uNK cells have important roles in development of implantation sites [5, 6]. Morphological studies of implantation sites from mice deleted for genes involved in one NK cell activation pathway (interleukin [IL]-12 and Stat-4) support the decidua and its vessels as targets of uNK cell functions [8], but neither those functions nor their regulation have been defined.
Cytotoxicity and/or cytokine release, particularly interferon (IFN)-, are the postulated major functions of NK cells [10, 11]. Previous in vitro [1, 12] and in vivo [1, 13] studies have suggested that cytotoxicity is not a major function of uNK cells during normal murine pregnancy. IFN- mRNA and protein have been reported within implantation sites and uNK cells [3, 14–16]. In the pregnant mouse uterus, uNK cells, macrophages, and decidual and placental cells express high levels of IFN- receptors [17]. Thus, both autocrine and paracrine IFN- signaling may be present in normal uteroplacental tissues.
IFN- regulates more than 200 genes in a wide variety of cells and tissues. Apoptosis, mRNA processing, transcriptional regulation, and major histocompatibility complex (MHC) gene expression are among the pathways influenced by IFN- [18]. IFN- is not essential for fetal survival in view of the fact that mice ablated for either IFN- or IFN-R are viable and give offspring as homozygous young mating pairs [19, 20]. It is widely held, however, that IFN- at implantation sites is detrimental to or incompatible with gestational success [21, 22].
The present studies were undertaken to determine whether abnormalities seen in uNK cell-depleted mice are independent of or dependent upon IFN- signaling. Three studies were conducted. First, IFN- was quantified at the mesometrial side of implantation sites on different days of gestation in four strains of mice, two immune competent (CD1 and BALB/c) and two immune deficient (tg26 and SCID). Second, pregnancy outcome and implantation site morphology were compared in IFN--/- and IFN-R-/- mice and their congenic partners. Third, the ability of fetally derived IFN- to support normal implantation site development in IFN--/- mothers was assessed.
These studies indicate that, during pregnancy, mesometrial IFN- peaks at Day 10 of gestation and is largely a maternal product, produced by uNK cells. Maternal cells other than NK and T cells are minor producers. Further, many of the pathological features seen in uNK-deficient mice were observed in IFN--/- and IFN-R-/- mice, suggesting that many of the contributions made by uNK cells to normal pregnancy are mediated through IFN- signaling.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Seven-week-old IFN--/-, IFN-R-/-, inbred BALB/c, and 129/SvJ mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Random-bred CD1 mice were obtained from Charles River Laboratories (St. Constant, PQ, Canada). Transgenic tg26 and outbred scid/scid (SCID) mice were bred at the University of Guelph (OMAFRA Isolation Unit, Guelph, ON, Canada). Female mice of all strains were syngeneically mated. In addition, some IFN--/- females were mated to BALB/c (IFN-+/+) males to obtain IFN-+/- fetuses, while others were mated to IFN--/- males and killed on Day 3 of gestation for recovery of blastocysts. These blastocysts were transferred to CD1 females mated 2 days earlier by vasectomized CD1 males as previously described [5]. At specific days of gestation (counting the copulation plug day as Day 0), mice were killed by CO2 and subsequent cervical dislocation. Uterine horns were dissected and examined for fetal viability (implant size and color), and then implantation sites were processed for study.
Tissue Acquisition for IFN- Quantification
To quantify local concentrations of IFN- at the sites where uNK cells are found, mesometrial decidua (gestation days 6–8) or lymphoid aggregates (gestation days 10–13, 16) representing the metrial glands were dissected using fine curved scissors. These tissues are not expected to contain fetal cells. Mesometrial sides of nonpregnant uteri were dissected to compare the levels of IFN- to those of the pregnant mice. Dissected samples were pooled by litter in 1.5-ml microcentrifuge tubes containing 100-µl RPMI 1640 medium with 10% fetal calf serum (FCS) and immediately homogenized by micropestle (Fisher Scientific, Whitby, ON, Canada). To quantitate IFN- concentrations contributed by the fetoplacental units of IFN-+/+ and IFN-+/- conceptuses, the fetal fluids, fetus, and placenta from each implantation site were collected separately at Days 12 and 14 of gestation for analysis. The fetus and placenta were homogenized as above. Samples were centrifuged at 800 x g for 5 min at 4°C. The supernatants were collected and stored at -20°C until analyzed for IFN- by ELISA. The amount of IFN- detected in each component (fluids, fetus, placenta) were summed to give the value of IFN- per fetoplacental unit. The IFN- for each fetoplacental unit was averaged with values obtained from all littermates to provide the mean IFN- contributed to the implantation site per conceptus.
ELISA Quantification of IFN-
Supernatants from homogenized mesometrial decidua or the mesometrial lymphoid aggregates were assayed for IFN- by ELISA using paired capture and biotinylated detection antibodies purchased from Pharmingen (Mississauga, ON, Canada). Briefly, ELISA plates (Dynex Technologies Inc., Chantilly, VA) were coated with 50 µl/well of 1 µg/ml of capture antibody (R4-6A2 rat anti-mouse IFN- antibody) in coating buffer (0.1 M Na2HPO4) overnight at 4°C. Coated plates were washed with PBS/Tween 20 (PBST) and then blocked with 10% FCS for 30 min at room temperature (RT). Titrated samples and recombinant mouse IFN- standards (Sigma, Oakville, Canada) were added to triplicate wells (100 µl/well) and incubated overnight at 4°C. The plates were washed four times with PBST and incubated with 50 µl of 0.5 µg/ml of biotinylated detection antibody (XMG1.2 rat anti-mouse IFN- antibody) at RT. After 60 min, the plates were washed six times with PBST and incubated with 100 µl of a 1:3000 dilution of horseradish peroxidase avidin D (Vector Laboratories, Burlingame, CA) at RT for 30 min and then washed eight times with PBST. Substrate (100 µl; 2.2'-azino [3-ethylbenzthiazoline-6-sulfonic acid], 1 mg/ml; 0.003% H2O2; 0.1 M citric acid) was added to each well. After 60 min at RT, absorbance was measured at a wavelength of 405 nm. IFN- concentrations were determined against a standard curve, constructed in each assay by serially diluting recombinant mouse IFN-. The minimum limit of detection of the assay was 0.01 U/ml. To calculate U/implantation site, the total units of IFN- in the sample were correlated to the final volumes and protein concentrations in each sample and then divided by the number of implant sites in the litter that had been pooled to produced the sample.
Morphological Analysis of Implantation Sites
Time course-collected samples of implantation sites were available from homozygously mated IFN--/-, IFN-R-/-, BALB/c, and 129/SvJ between Days 8 and 18 of gestation. Implantation sites from second pregnancy of IFN--/- and BALB/c were available on Days 10, 12, 14, and 16. The implantation sites were fixed in Bouin's fixative (Fisher Scientific) overnight and processed into paraffin. Two to five implantation sites from each pregnant mouse were serially sectioned at 7 µm and stained using a standard protocol for periodic acid-Schiff (PAS) to identify glycogen-containing cells. All stained slides were examined by light microscopy. The center section of each serially sectioned implantation site was identified and, for each implantation site, 4 median tissue sections on each side of the central section were also analyzed (n = 9). Sections were at least 42 µm apart to avoid duplicate counting of individual uNK cells. The number of uNK cells per square millimeter in the metrial gland and in the decidua basalis was counted at x500 magnification using an eyepiece micrometer grid of 1 mm2. The circular smooth muscle was used as the dividing line between the metrial gland and decidua basalis. The areas of the metrial gland, decidua, and placenta, as well as wall:lumen ratios for the large arterioles thought to be analogous to human spiral arterioles, were image analyzed using the Northern Exposure system 2.6a (Imagexperts, Inc., Hollywood, CA).
Terminal Deoxynucleotidyl Transferase (TDT) Assay (TUNEL Assay)
Analysis of DNA strand breakage was performed with a previously described modification of the TUNEL assay [23]. Briefly, 7-µm paraffin sections were cut and fixed overnight on positively charged slides (Superfrost Plus; Fisher Scientific). Slides were incubated with proteinase K (10 µg/ml) and then treated with 1% H2O2 for endogenous peroxidase inactivation. About 50 µl of terminal transferase reaction from Boehringer Mannheim (Mannheim, Germany) (30 mM Trizma base, pH 7.2, 140 mM sodium cacodylate, 1 mM cobalt chloride, 0.3 U/µl TDT, and 10 mM of biotinylated dUTP) was added to each section, which was then incubated in a humid atmosphere at 37°C for 60 min. The sections were covered with 2% BSA for 10 min at RT and then covered with 100 µl of diluted (1:5000) extra-avidin peroxidase conjugate (Zymed Laboratories, South San Francisco, CA) for 30 min at RT. Slides were then incubated with the substrate, 3,3'-diaminobenzidine tetrahydrochloride. Sections were stained with PAS as a counterstain. Positive and negative controls were used in each assay. Tissues from implantation sites of homozygously mated tg26 females, collected in previous work [5,6], were also examined by TUNEL.
Sample Preparation for Electron Microscopy
Mesometrial decidua and metrial glands from implantation sites of IFN--/-, IFN-R-/-, and their congenic partners were dissected on Day 12 of gestation and fixed in immersion fixative (2.5% glutaraldehyde and 2.5% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4) for 3 h. Fixed tissues were washed with 0.1 M phosphate buffer three times (15 min each) and then postfixed for 90 min in 1.5% osmium tetroxide (OsO4) in 0.1 M phosphate buffer. Fixed tissues were dehydrated in ascending grades of ethanol to 100% and embedded in Jembed 812 resin (Fisher Scientific). Semithin sections (1 µm) were cut and stained with toluidine blue for preview. Ultrathin sections of selected samples were cut using a Sorvall-MT-2B ultramicrotome (DuPont Instruments, Newtown, CT) and stained with uranyl acetate and lead citrate. The stained sections were examined with JEOL-100S (Peabody, MA) transmission electron microscope (TEM) at 100 KV. Several fetuses of each genotype were examined from each of two mothers.
Statistical Analysis
Fetal resorption rates were compared by chi-square with Yate's correction. The uNK cell numbers at implantation sites and cross-sectional areas present in metrial glands and decidua basalis were compared between experimental and control groups by ANOVA using Statistical Analysis Systems computer software program (Cary, NC). A p value of less than 0.05 was considered a significant statistical difference.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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in Pregnant Mouse Uteri
To assess the mesometrial concentration of IFN- in implantation sites, IFN- was measured in homogenates of freshly dissected mesometrial tissue. Figure 1 compares the levels of IFN- found. The pattern of IFN- detection was similar in the immune-competent random-bred and inbred strains and in a T and B cell-deficient strain. In each of these, IFN- was detected on every gestational day examined, and the levels per implantation site rose from Day 6 to a peak at Day 10 of gestation. A dramatic drop occurred at Day 11 of gestation to a level that was somewhat variable between strains. Unexpectedly, IFN- was detected in the mesometrial region of tg26 mice that lack NK and T cells. The levels were lower than those seen in NK+ mice and were stable across the gestation days available for study. The homogenates from the mesometrial side of implantation sites from IFN-/- females (congenic to BALB/c) were unreactive in the assays and represent a negative specificity control. Samples from nonpregnant uteri were not significantly different from the negative controls (shown as Day -1 of gestation in Fig. 1).
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Assessment of Fetal Viability and Histological Appearance of Implantation Sites
During tissue collection from IFN--/- mice, a high fetal resorption rate was noted (Table 1). This was not found in the congenic strain. The first deaths, noted at Day 8, involved several fetuses in each litter. After Day 12 of gestation, no additional fetuses appeared to be lost. Jackson Laboratories, the supplier, indicated that this gene-deleted strain has small litters only during first pregnancy. This was quantified by permitting females to give birth, remating them, and killing them during second pregnancy. These females had smaller first litters compared to their congenic strain, but there were no differences in the litter sizes during second pregnancy (Table 1). IFN-R-/- mice did not show fetal loss during first pregnancy and were not studied during second pregnancies (Table 1). Only viable implantation sites from first and second pregnancies were used for histology.
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At Day 8 of gestation, implantation sites from IFN--/- and IFN-R-/- had features similar to those of congenic controls and were assessed as normal (Fig. 2). Uterine NK cells were present. Figure 3 compares uNK cell enumeration in midgestation implantation sites from both IFN--/- and IFN-R-/- to that for their congenic partners. Higher numbers of uNK cells were present at the metrial glands of both gene-ablated strains after Day 10 of gestation. By TUNEL, IFN--/- and IFN-R-/- mice had a lower frequency of apoptotic uNK cells in the metrial gland (30% on Day 14 of gestation) than found in normal mice (73% in this study in controls and 80% in published literature [23]). This reduced cell death could account for the higher numbers of uNK cells in the gene-ablated mice. Uterine NK cells, although abundant at implantation sites, failed to become heavily granulated (Fig. 4). The unusual appearance of these cells was established ultrastructurally at Day 12 of gestation (Fig. 4F). Uterine NK cells from both IFN--/- and IFN-R-/- had granules of variable electron density with irregular shapes and abnormal cap formation compared to cells from normal mice (congenic data not shown, [24]). The Golgi apparatus was poorly developed, which could contribute to poor granule formation. The rough endoplasmic reticulum was disorganized within the cytoplasm. Numerous mitochondria were observed, suggesting high metabolic activity.
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In IFN--/- mice, after Day 10 of gestation, the intercellular composition of the decidua changed to suggest accumulations of fluid and extracellular matrix. Areas of necrosis were present, and a lack of decidual cellularity was apparent as compared to time-matched BALB/c decidua (Fig. 2C). The major decidual arterioles had thicker walls than seen in the control BALB/c strain (Fig. 2, E and G). Table 2 compares the mean decidual vessel wall to lumen cross-sectional areas between IFN--/- mice and their congenic controls. No significant nuclear fragmentation was present in mesometrial decidual cells at Days 8 and 10 of gestation but was found on Days 12 and 14. There were no histologic differences between viable fetuses gestating in primiparous versus multiparous IFN--/-.
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Implantation sites of IFN-R-/- mice had pathology similar to those in IFN--/- mice, but decidual lesions were more severe. In IFN-R-/- pregnancies, the entire mesometrial decidua became progressively necrotic and was lost. By Day 14, only a thin layer remained in the tissue cross sections (Fig. 2, D and F). Decidua necrosis was confirmed using the TUNEL assay (Fig. 2, H and I). Decidua from congenic 129/SvJ mice was normal, with healthy decidual cells (Fig. 2, B and H). Area measurements of the decidua and metrial gland regions revealed that IFN-R-/- mice had reduced decidual tissue but larger metrial glands than their congenic controls (Fig. 5).
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Acellularity of decidua is present in tg26 females (uNK-), but this has not been linked to decidual cell death. To determine whether death of decidual cells was also occurring in tg26 females, the TUNEL assay was employed on tissue sections from Days 12 and 14 of gestation. No evidence was obtained for elevated decidual cell death above that observed in immune-competent control tissue sections (data not shown). Thus, absolute loss of signaling via IFN- pathway had a more severe effect on the decidua than did the combined loss of uNK cells and peripheral NK and T cells.
Assessment of Fetally Contributed IFN- to Implantation Site Normalcy
To determine whether fetally derived IFN- contributes to the normalcy of the maternal uterine environment in midgestation, IFN--/- females were mated to BALB/c (IFN-+/+) males, yielding IFN-+/- fetuses. At Days 12 and 14 of gestation, each conceptus produced 2.2 and 2.6 U of IFN-, respectively. Implantation sites containing heterozygote fetuses on these days (Fig. 6A) had features similar to those of IFN--/- females carrying fetuses unable to produce IFN- (Fig. 2C). These results indicate that no rescue of the phenotype had occurred. In the reciprocal experiment, IFN--/- blastocysts were transferred to pseudopregnant CD1. This removed IFN- production by wild-type fetoplacental units, which was measured at 2.8 and 3.1 U/fetoplacental unit at Days 12 and 14 of gestation. These implantation sites and the uNK cells found and enumerated there were similar to those in CD1 females mated by CD1 males (Fig. 6B). Both breeding strategies suggest that maternal IFN- has the dominant role in sustaining decidual health.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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within implantation sites. The results suggest that IFN- is a major product of murine uNK cells during pregnancy and that IFN--mediated signaling, from uNK cells as well as other cells, is essential for normal maintenance of maternal tissues in implantation sites during midgestation.
To understand the role of IFN- during murine pregnancy, it was first necessary to establish the concentration of IFN- in the region of the pregnant uterus where uNK cells are found. Murine uNK cells are not available as cell lines or clones, and since they are large and fragile, they rupture during normal cell suspension procedures. The technique for collection of migratory uNK cells from mesometrial tissue explants does not maintain nuclear integrity [25]. Thus, immediate homogenization of freshly dissected mesometrial tissue from implantation sites was used to obtain comparable samples that would mimic as closely as possible the in vivo situation. Neither these homogenates nor supernatants from migrating uNK cell cultures could be used in the WEHI 279 bioassay for IFN- since the target cells were immediately killed (data not shown), giving false-positive results for growth inhibition of the target cells. The dual-antibody ELISA worked reliably with the mixed tissue homogenate.
The most important result from the IFN- time-course study in normal mice was that levels of IFN- were not constant but instead gradually increased from Days 6 to 10 of gestation and then dropped. Uterine NK cells are gradually increasing in number and in granularity between Days 6 and 10 of gestation [1, 3, 24]. To determine which cells were the source of the early rise and Day 10 peak of mesometrial IFN-, the cytokine was assayed in mesometrial regions from immune-deficient mice. SCID mice, which are similar in numbers and developmental time course of uNK cells to normal mice, had mean levels of IFN- comparable to those of immune-competent mice that followed a similar time-course pattern, indicating that cells present in SCID mice are the sources of mesometrial IFN- during normal pregnancy. These cells were expected to be uNK cells. However, IFN- was constantly found in tg26 implantation sites at a relatively constant, low concentration. Since uNK cell frequency in tg26 mice is 1% that of SCID mice (range of 0–3% [5]), it is unlikely that uNK cells were responsible for the IFN- detected, as the IFN- values were 12–20% of the peak values seen per implantation site at Day 10 in the NK+ strains. Further, the production of a constant concentration of IFN- in tg26 implicates another IFN--producing cell type. Platt et al. [15, 16], using immunohistology and in situ hybridization, also found that uNK produce IFN- in the gestational uterus. In the mesometrial location, macrophages, neutrophils, or decidual cells are all possible nonlymphoid sources for the detected cytokine [16, 26, 27]. The level of IFN- recorded in tg26 was not an artifact, since the assay employed was negative when homogenates from the same tissues collected from IFN--/- mice were tested.
Several anomalies are found in the pregnant uteri of uNK-deficient mice [5, 8], becoming evident by light microscopy at Day 10 of gestation [5] and ultrastructurally by Day 8 of gestation (unpublished results). These include no uNK cells or metrial gland. In IFN--/- and IFN-R-/- mice, uNK cells and metrial glands both differentiated but were anomalous. Uterine NK cells had fewer, poorly developed granules with abnormal ultrastructure. This is a predictable result, since IFN- regulates new mRNA synthesis and the synthesis and assembly of ribosomes and their component molecules [28, 29]. Uterine NK-deficient mice have vascular pathology in the decidual arterioles; and the decidua itself is either relatively acellular or edematous, but the decidua cells do not show nuclear fragmentation, indicative of cell death. IFN--/- and IFN-R-/- mice had similar implantation site morphology (Fig. 2) but more extreme decidual failure. The overt necrosis of the decidua seen in these two strains suggests that in NK cell-deficient mice, the undefined cell population that produces constant levels of IFN- was cytoprotective for the mesometrial decidual cells. Loss of decidua in the last trimester of gestation had no apparent effect on fetal survival. Fetal loss was not seen in IFN-R-/- females or in second pregnancy in IFN--/- females. The losses seen in first pregnancy of IFN--/- females occurred prior to onset of nuclear fragmentation in decidual cells. The presence of decidual vessel and tissue anomalies, in IFN--/- and IFN-R-/- mice, indicates that uNK cells present in these mice do not function normally when IFN- or its signaling pathway is lacking. The related decidual phenotypes in the four known uNK cell-deficient mice and the two IFN- pathway-deficient mice examined in this study strongly suggest that the major functions of uNK cells in the pregnant uterus are mediated through IFN--regulated genes [30]. The basis for the more severe decidual phenotype seen in the IFN-R-/- is not known. Perhaps, owing to the IFN- signaling deficit, MHC expression on decidual cells has dropped below levels necessary to engage the killer-inhibitory receptors of NK cells and the uNK cells have become lytically active and are destroying the decidual cells [31–33]. A weakness of this idea is that ultrastructurally, the granules of uNK cells where perforin and serine esterases localize are poorly developed in IFN--/- and IFN-R-/- mice [1, 7, 13]. Others studying these two strains in pathogen challenge studies, as well as mice deleted in IFN-Rß [34] and signal transducer and activators of transcription (Stat)-1 (the transcription factor mediating IFN- signaling) [29, 35], report related but distinctive phenotypes [34]. IFN-R-/- mice are able to generate T helper 1 (Th1) cells whereas IFN--/- do not [34]. The Stat-1 pathway can also be triggered by other receptors such as the lipopolysaccharide receptor to mimic IFN--like signals [36]. Both Janus kinase (JAK)-1 and JAK-2 are utilized by the IFN- receptors to form the Stat-1 docking site. IFN-/ß, interleukins 2, 3, 4, 6, 7, 9, 10, 12, 15, granulocyte-macrophage-colony stimulating factor, and prolactin use JAK-1 or -2 [29], making many compensatory pathways possible that might be differentially used in the cytokine-deleted and the receptor-deleted animals. It is possible that compensation through one of these pathways becomes successfully established in first pregnancy in IFN--/- females and is sufficient to promote survival of larger litters in subsequent gestations.
To determine whether the anomalies seen in IFN--/- mice were due to the loss of maternally or fetally derived IFN-, implantation sites from IFN--/- females mated by BALB/c males (congenic controls) and CD1 females that received IFN--/- blastocysts were studied. Production of IFN- by murine conceptuses commences at gestation Days 6–7 [14, 37], and significant levels are produced by IFN-+/- fetuses [38]. Since the IFN--competent fetuses did not reverse the gestational anomalies of IFN--/- females, and IFN--/- fetuses had no effect on CD1 recipient implantation sites, fetally derived IFN- must not be produced at sufficient concentrations or delivered to the maternal environment and is unable to compensate for maternally derived IFN- during pregnancy.
The immunology of pregnancy, like other areas of immunology, has been heavily influenced by Th1/Th2 cytokine paradigm. Wegmann et al. proposed that successful pregnancy is a Th2-dominated (e.g., IL-4 and IL-10) phenomenon [21], while Raghupathy proposed that Th1 cytokines, particularly IFN-, are incompatible with successful pregnancy [22]. These conclusions are supported by elevation of IFN- in the serum of women with recurrent spontaneous abortion and in vitro and in vivo studies in resorption-prone mice [39, 40]. However, it is unclear whether the Th1-type cytokines, particularly IFN-, generated the problems or are secondary responses to failure of other mechanisms. Published data are beginning to challenge the Th1/Th2 paradigm [41, 42] and show greater complexities in vivo. Numbers of circulating IFN--secreting cells are reported to be elevated in all three trimesters of human pregnancy [43], supporting our findings of IFN- production at normal murine implantation sites. Thus, IFN- at concentrations under 10 U/mouse implantation site is not only nondetrimental to pregnancy but is essential for normalcy of implantation sites and maintenance of decidual cell viability. In implantation sites, IFN- is largely derived from uNK cells, and it may be the major regulatory pathway by which uNK cells contribute to gestation.
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ACKNOWLEDGMENTS |
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26 mice; and Mr. Andrew Moore (Laboratory Services Division, University of Guelph) for providing access to the Northern Exposure Image Analysis System. |
FOOTNOTES |
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2 Correspondence. FAX: 519 767 1450; aashkar{at}ovcnet.uoguelph.ca
Accepted: March 25, 1999.
Received: August 6, 1998.
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