BIOLOGY OF REPRODUCTION 56, 169-179 (1997)
Absence of Natural Killer Cells during Murine Pregnancy Is Associated with Reproductive Compromise in TgE26 Mice Marie-Josee Guimond, and B. Anne Croy 3
23 ,
Jeffrey A. Luross, 3 Baoping Wang, 4 Cox Terhorst, 4 Susan Danial, 3
Department of Biomedical Sciences, 3 University of Guelph, Guelph, Ontario, Canada NIG 2W1 Division of Immunology,4 Beth Israel Hospital, Harvard Medical School, Boston, Massachusetts 02215 ABSTRACT
and during birth, all remaining GMG cells are shed with the placenta as components of the afterbirth [1, 12, 13]. The major functions of these pregnancy-associated lymphocytes are poorly defined, but the cells are known to produce cytokines [2]. One approach towards understanding the functions of GMG cells is to deplete them in vivo and observe the consequences on pregnancy. If the depletion of GMG cells leads to changes in the reproductive process, an important role for GMG cells during pregnancy may be established. Attempts to deplete mature GMG cells from the uterus during pregnancy have been reported using repeated infusion of mated mice with antibodies against NK 1.1 (on Day 10, Days 6 and 10, or Days 2, 6, and 10 of pregnancy) or asialo GM1 (on Days 1, 5, and 9 of pregnancy) [6]. These experiments demonstrated depletion in splenic NK cells, but no depletion in the numbers of mature uterine GMG cells [6]. Pregnancies in treated animals were normal. An attempt to deplete GMG cells in female mice through repeated breedings (> 10 litters) has been reported to reduce GMG cell numbers by one third; however, this reduction in cell frequency did not result in any changes in placental area or litter size [14]. Genetic depletion has been successful for understanding lineage relationships and many of the functions of lymphohematopoietic cells. However, there are no known spontaneous mutations in mice wherein the NK cell lineage is deleted. The beige (bg) mutation decreases lytic ability of murine NK cells but does not alter the frequency of NK cells in either the peripheral blood [15] or uterus [16]. In culture, cytokine production by GMG cells from beige mice cannot be distinguished from that by GMG cells from normal mice [17]. Newer strains of mice lacking the cytokine interleukin (IL)-2 or the IL-2 receptor complex might be expected to have a deficit in GMG cells. IL-2R-y knockout mice are NK- and T-cell-depleted with a greater reduction in the NK cell lineage; however, because of the position of the IL-2Ry gene on the X chromosome, analysis of the depleted phenotype has been reported only in males [18]. Other transgenic mice lacking NK cells in spleen and bone marrow have been reported [18-20]. One series of transgenic mice carrying varying doses of the human CD3E gene lacks NK and T cells beginning in fetal life [20]; these mice include the TgE26 and Tg2978 strains. To extend the characterization of GMG cell function over the course of gestation, we examined pregnant mice depleted of NK cells by either antibody treatment or genetic manipulation.
Strategies of cell depletion were pursued to extend understanding of the functions of natural killer (NK) cell-like large granulated lymphocytes found in the rodent uterus during pregnancy. Repeated infusions of antibody to Ly-49G2, a surface marker thought to be expressed by the progenitor forms of these cells, removed Ly-49G2+ cells from the virgin but not the pregnant uterus. Large granulated uterine lymphocytes also differentiated during pregnancy in transgenic mice that carried a deletion in the IL-2 gene. This cell population was absent in two strains of mice, p56'ck-'ck-.L-2R-/IL-2RI
and TgE26. Im-
plantation sites in both of these strains had histopathological anomalies in the zone of decidualization. In TgE26 mice, a sudden onset of fetal loss began at Day 10 of gestation. Fetal death was associated with progressive changes in the maternal uterine arterioles, suggestive of localized arteriosclerosis associated with hypertension. TgE26 females carried immune-competent fetuses to term, apparently through preventive or compensatory mechanisms that may modify the uterine vasculature after the onset of vascular pathology. These studies are the first to suggest a vital role for large granulated lymphocytes in the promotion of fetal survival and pregnancy success. INTRODUCTION Microanatomists noted the differentiation of a specialized region on the mesometrial side of each placenta in the pregnant murine uterus, a region called the mesometrial triangle. Up to 20% of the cells within a fully developed mesometrial triangle (Day 10 of gestation and later) are large, granulated lymphocytes, known widely as granulated metrial gland (GMG) cells [1, 2]. Genetic studies have indicated that GMG cells are neither B nor T lymphocytes; immunophenotyping has supported their assignment to the natural killer (NK) cell lineage. Agranular precursors of GMG cells have been described at Day 6 of pregnancy [1, 3] that express Ly-49G2 (formerly known as LGL-1) [4, 5], but lack asialo GM1 [1, 6]. By midgestation (term is 19-21 days in mice), GMG cells become large, heavily granulated cells that express an NK cell-like surface phenotype of CD3-, Thy-1 +, asialo GM1 +, NK1.1 +, 4H12+ [7-10], and IL-2Rot3y+ (M. van den Heuvel and B.A. Croy, personal communication). The relationship of the GMG cells to NK cells in other sites has not been fully described. It has been postulated that GMG cells are typical NK cells [10], a specialized subset of NK cells [2], or uterine cells unrelated to other NK cells [11]. As term approaches, GMG cells disappear from the uterus by necrosis,
MATERIALS AND METHODS
Accepted August 20, 1996. Received June 21, 1996. 'Supported by the Natural Sciences and Engineering Research Council of Canada and the Ontario Ministry of Agriculture, Food and Rural Affairs. 2Correspondence. FAX: (519) 767-1450; e-mail:
[email protected]
Animals Random-bred CD1 mice were purchased from Charles River Laboratories (St. Constant, PQ, Canada). Inbred CBA/J and C3H/HeJ (both H-2k) mice were purchased 169
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from Jackson Laboratories (Bar Harbor, ME). Young adult homozygous double mutant p56Ik /lck .IL-2R3-/ IL-2R3- mice [21, 22] and homozygous IL-2-/- mice [23] were generously donated by Drs. Tak W. Mak and Haruhiko Suzuki from the Amgen Institute, University of Toronto (Toronto, ON, Canada), and used for timed matings in Guelph, ON, Canada. Timed matings of IL-2-/-.132m-/ - mice were conducted in Boston, MA, and fixed uteri were shipped to Guelph. A breeding colony of transgenic TgE26+/+ and Tg2978+/- mice [20] was maintained in the barrier-sustained environment of the Isolation Unit at the University of Guelph. TgE26+/+ (H-2k) and Tg2978+/ - (H-2 undefined) mice were developed by pronuclear injection of related human CD3E transgenes. TgE26+/+ and Tg2978+/- mice were weighed at weaning (3 wk of age) and at adulthood (7 wk of age). Three estimates of weight for each mouse were recorded, and the mean was calculated; gender was recorded for each mouse. The studies were carried out in accordance with the principles and procedures described in the guidelines for the care and use of laboratory animals as approved by the respective institutions. Estrous females were selected for mating to syngeneic males. The morning that a vaginal copulation plug was detected was called Day 0.5 of pregnancy. At specified times, some mice were anesthetized with 0.4-0.6 ml of tribromoethanol anesthesia (10 mg/ml) [24] and used for surgery or perfusion (10 ml of 4% paraformaldehyde) and tissue collection. Other mice were killed using CO 2 followed by cervical dislocation before tissue collection. Embryo Transfers Female mice were superovulated by an i.p. injection of 5 IU eCG (Calbiochem-Novabiochem, La Jolla, CA), followed 48 h later by an i.p. injection of 5 IU hCG (Sigma Chemical Co., St. Louis, MO). Immediately after the hCG injection, females were placed in cages with fertile males and examined the following morning for a copulation plug. Mated mice were assumed to be pregnant and were used as embryo donors. Other estrous females were paired with vasectomized males and examined the following morning for a vaginal plug. These pseudopregnant mice were used as embryo transfer recipients. Embryo transfers were performed with the embryo donors at Day 3.5 of gestation and the recipients at Day 2.5 of gestation. All morulae and blastocysts deemed viable upon microscopic inspection were transferred by an experienced individual (B.A.C.). M2 medium [24] was used for flushing of the uteri and embryo holding. Tissue Collection For histological analysis, one or more of the following tissues were dissected from mice: the thoracic pluck consisting of heart, aorta, and lung; the carotid arteries; kidneys; liver; spleen; ovaries; and uterus. Pregnant uteri were assessed for conceptus viability as indicated by implant size and color. These uteri were processed as individual implantation sites. For paraffin processing, tissues from the nonperfused animals were placed in either Bouin's fixative or 10% phosphate-buffered formalin for 3-6 h. Perfused tissues were placed into 4% paraformaldehyde in PBS at 4°C overnight. All fixed tissues were then stored in 70% ethanol until further processing for paraffin embedding, sectioning (thickness of 6 tm), and staining using periodic acid Schiff (PAS) [25] or hematoxylin and eosin (H&E) reagents. For
immunohistochemistry, tissues were collected and placed into O.C.T. embedding compound (Miles Laboratories, Elkhart, IN) and stored at -70°C until cryostat sectioning. Time-matched pregnant uteri from syngeneically mated CDI and CBA/J mice were used as normal controls for the histological studies. Mature GMG cells were recognized by the reactivity of their granules with PAS. The Northern Exposure System 2.6a (Imagexperts Inc., Hollywood, CA) running in Microsoft Windows 3.1 (Microsoft Corp., Redmond, WA) was used to quantify the frequency of PAS-reactive cells and areas of placentae. Antibodies The monoclonal antibody (mAb) rat anti-mouse Ly-49G2 (IgG2a) was generously provided by Dr. L.H. Mason (National Cancer Institute, Frederick, MD). For immunohistochemistry, Ly-49G2 was used at 1:500 in PBS and was detected by the secondary reagent TRITC goat anti-rat IgG (Cedarlane Laboratories, Hornby, ON, Canada) used at 1:500 in PBS. Rat IgG2a (Pharmingen, San Diego, CA) was used (1:500 in PBS) as the negative isotype control reagent. For in vivo treatments, Ly-49G2 was given in doses of 250 pl (19.9 mg/ml) per day. In Vivo Depletion of Ly-49G2+ Cells Virgin, nonpregnant, and pregnant CDI mice were treated i.p. with either Ly-49G2 antibody or PBS. To establish the treatment protocol, a mouse received 250 1l of antibody daily for 1, 2, 3, or 4 days and was killed 24 h after receiving the final treatment. Pregnant CDI mice (n = 4) were given 250 pIl of anti-Ly-49G2 daily for 4 days over the implantation period (Days 1.5-4.5 of gestation) whereas control matched mice (n = 2) received 250 pl of PBS. The pregnant uteri were studied at Days 7 or 12 of gestation. Immunohistochemistry for Detection of Ly-49G2-Reactive Cells Six-micrometer cryostat sections were prepared from the O.C.T.-embedded tissues and mounted onto slides coated with 3-aminopropyltriethoxy-silane (APTEX; Sigma Chemical Co.). All slides were fixed using 2% phosphatebuffered paraformaldehyde (pH 7.4) for 10 min at 4°C. Indirect immunohistochemistry was performed using standard methods [26]. For nonpregnant uteri, a minimum of 20 sections were examined per mouse. For pregnant animals, 2 implantation sites were studied from each pregnancy with a minimum of 10 sections per implantation site. Specimens from each animal were studied in at least two independent experiments. Slides were viewed using a Leitz Aristoplan epifluorescence photomicroscope. In the Ly-49G2 infusion studies, cells were enumerated at 400x magnification using a 24-mm 2 grid. The term "frequency" is used as a comparative measure of the number of cells per grid in different samples. Fluorescence was not observed in the absence of the primary antibody. Each experiment was repeated at least twice. Statistical Analysis Means, ranges, standard deviations, p values and paired t-tests were performed using the computer software program Microsoft Excel for Windows 5.0 (Microsoft Corp.).
GESTATION IN NK CELL-DEFICIENT MICE TABLE 1. Treatment of nonpregnant and pregnant CD1 mice with Ly-49G2 antibody. Day of gestation NPI NP NP NP NP 7 7 7 12 12 12
Treatment (n = 2) 1.0 ml PBS 0.25 ml Ly-49G2 0.50 ml Ly-49G2 0.75 ml Ly-49G2 1.0 ml Ly-49G2 nil 1.0 ml Ly-49G2 1.0 ml Ly-49G2 nil 1.0 ml Ly-49G2 1.0 ml Ly-49G2
Mean no. of PAS+ cells
Mean no. of Ly-49G2+ cellsb
N/A d N/A N/A N/A N/A 12.7 - 2.3 10.4 3.4 11.8 + 4.9 139.7 17.3 154.1 + 21.6 + 137.0 19.8
24.0 + 6.8 17.6 + 4.1 5.0 1.7* 6.4 + 1.6* 4.4 + 1.5* 10.7 _ 1.3 10.1 _ 2.4 13.5 4.4 N/A N/A N/A
PAS-reactive cells were counted using one 1-mm 2 grid/section, at 500x for the Day 7 implants and at 250x for the Day 12 implants. 2 b Ly-49G2+ cells were counted using one 24-mm grid/section, at 400x. c NP = nonpregnant. d N/A = not applicable; the Ly-49G2 surface antigen is lost by Day 12 of gestation. * Significantly different from the control animals but not from each other (p < 0.001). a
RESULTS
Antibody Depletion of Uterine Ly-49G2 + Cells Antibody-mediated depletion of uterine GMG cells was attempted using an mAb directed against the putative Ly-49G2+ precursor cell. To establish an effective dose for uterine depletion of Ly-49G2+ cells, four nonpregnant mice each received a different dose of anti-Ly-49G2 antibody (Table 1). Mice receiving more than one dose showed a 73% or higher depletion in uterine Ly-49G2+ cell frequency when compared to the PBS control. From this study, the longest treatment regimen with the highest cumulative dose (1 ml) was chosen as the optimal protocol for treatment of mated mice over the periimplantation period. Only one fetal resorption site was observed among 79 implantation sites in the groups of pregnant females studied at Days 7 and 12 of gestation (n = 6). Pregnancies in mAbtreated females appeared normal, as were the numbers of implantation sites compared to those of PBS-treated controls. Histologically, neither Ly-49G2 immunohistochemistry (Day 7 implants), nor PAS staining (Day 7 and 12 implants) showed reduced GMG cell numbers (Table 1). Thus, the pregnant uterus was not depleted of mature GMG cells by repeated infusion of Ly-49G2 antibody, although the nonpregnant uterus appeared to lose the putative progenitor cells through an identical treatment. Genetic Depletion of GMG Cells from the Uterus To identify a murine depletion model for in vivo study of GMG cell function, we screened implantation sites collected from pregnant females from each of the following strains: IL-2-/-, IL-2-/-.3 2 m-/-, p561ck-Ack-.IL-2R3-/ IL-2RPand TgE26. At least two implantation sites were studied from each female on Day 14 of gestation. This time was chosen as the time when metrial gland development should be maximal. Figure 1, A-E, depicts typical, midsagittal, Day 14 sections of implantation sites from each mutant strain and from an inbred CBA/J implant. 1. IL-2-/- mouse. Two pregnant uteri were available for study containing 15 implantation sites. All implantation sites were assessed as viable by gross examination. Histo-
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logical examination of four implantation sites revealed morphologically normal placentae, mesometrial triangle development, and GMG cell differentiation, when compared to CBA/J placentae. No further studies were conducted with this strain. 2. IL-2-/-.,32m-/- mouse. Two pregnant uteri were available containing 12 implantation sites, of which five were resorbing. Histological examination of two viable implantation sites revealed a normal placenta with development of the metrial gland and differentiation of GMG cells. This strain, which was thought to have greater fecundity than the IL-2-/- knockout mouse (unpublished results), was not investigated further. 3. p56'ck-k- . IL-2R3 -/L--2R3- mouse. Four uteri were available for study containing 34 implantation sites. All implantation sites were deemed viable by gross examination. Histological examination of five implantation sites revealed a typical placenta, no metrial gland, and the virtual absence of GMG cells. The decidual region was deficient in typical decidual cells and appeared as a large area of adipose tissue. Since females of the parent strains were unavailable for study because of infertility, further studies would not have clarified whether the mechanisms accounting for the placental changes could be attributed directly to the loss of GMG cells. Thus, NK cell-deficient transgenic mice were assessed to substantiate this finding of decidual pathology in the absence of GMG cells. 4. TgE26 mouse. Two TgE26 uteri were examined on Day 14 of gestation. Fetal viability was extremely low; eight of 12 embryos (67%) were in late stages of resorption. In all four viable embryos, placental size was reduced although all trophoblast layers were present. The area of the placenta was 45% of the size of a Day 14 CBA/J placenta. There was no development of the mesometrial triangle area into a metrial gland. PAS staining revealed an almost complete absence of GMG cells. GMG cells were measured at 3% of the levels found in control CBA/J mice and were present only in the spongiotrophoblast region. Histological examination of the ovaries revealed normal corpora lutea without pathology (Fig. 1F). Furthermore, a total of 12 corpora lutea were found, corresponding with the litter size. Thus, ovarian changes did not appear to account for the loss of fetuses. The TgE26 mouse was chosen for extended study. Postnatal Reproductive Data Matings of homozygous TgE26 mice produced smaller litters (mean of 6 offspring per litter from 20 females, total 68 litters) than litters produced by matings of heterozygous Tg2978 mice, which have a similar genetic background and carry a related transgene (mean of 9 offspring per litter from 4 females, total 7 litters). Many TgE26 pups were small, and 11% of pups did not survive their first 24 h postpartum. Among the surviving pups, another 29% died before weaning. Thus, 40% of the TgE26 offspring born did not survive, and an average of 4 pups were weaned per litter. Figure 2 compares the weights of TgE26 mice to the weights of Tg2978 mice. At 3 wk of age, TgE26 pups weighed 29% less than age- and sex-matched Tg2978+/control mice (p < 0.001). Their smaller body weight persisted through adulthood, with the TgE26 mice weighing 27% less than control Tg2978+/- animals at 7 wk of age (p < 0.001).
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FIG. 1. Photomicrographs of midsagittal sections of Day 14 implantation sites collected from mutant and normal mice, stained with H&E. Implantation sites demonstrating typical placental (p) structure and presence of the metrial gland (mg) were seen in IL-2-/- mice (A) and IL-2-/ -. 32m-/- mice (B). C) The implantation sites from p561ck ,lc .IL-2R-/IL-2RPmice contained a typical placenta but lacked the metrial gland (-mg). Further, the decidual region (ad) was deficient in typical decidual cells and appeared as a large area of adipose tissue. Implantation sites from TgE26 mice (D) were smaller than those of control CBA/J mice (E) and lacked a metrial gland (-mg). F) Histologically, TgE26 ovaries collected on Day 14 of gestation were normal and contained corpora lutea (arrowheads). A-E) bar = 800 tIm, F) bar = 270 pim.
Prenatal Reproductive Data A time-course study of implantation sites from homozygous TgE26 was undertaken between Days 6 and 17 gestation (Fig. 3). Control groups were random-bred CD1 mice and inbred pregnancies from CBA/J and C3H/HeJ mice, both of which share an H-2k major histocompatibility complex (MHC) haplotype with noninbred TgE26 mice. TgE26 mice mated successfully: all copulation plugs resulted in pregnancies. TgE26 homozygous females had the same rate of ovulation (mean of 12 corpora lutea, n = 17 pairs of TgE26 ovaries) as CBA/J mice (mean of 13 corpora lutea, n = 10 pairs of CBA/J ovaries). From Day 6 to Day 8 of gestation, TgE26 mice had a slightly higher number of implants (mean number of implants in 8 TgE26 litters, 9.2; range 4-11; 100% viability) as compared to CBA/J mice (mean number of implants in 3 CBA/J litters, 7.3; range 68; 95.5% viability). However, after Day 9 of gestation, con-
ceptus viability declined to 71 % (mean number of implants in 15 TgE26 litters, 9.5; mean number of viable implants, 7.1; total range in litter sizes including both the viable and resorbing embryos, 4-14). In no case was an entire TgE26 pregnancy lost. Conceptus viability was 92.2% in immunecompetent, random-bred CD1 control litters (n = 23) and 80.3% in inbred control litters (CBA/J n = 9 and C3H/HeJ n = 5) between implantation and term. Pregnant heterozygous Tg2978 females were not available in sufficient numbers for a time-course analysis. Histological Analysis Implantation sites of viable TgE26 fetuses at Days 6, 7, 8, and 9 of gestation did not have features considered beyond those of normal variation (Fig. 4, A and B). From Day 10 of gestation, various histological changes were observed (Figs. 4 and 5). The most dramatic change was the
GESTATION IN NK CELL-DEFICIENT MICE reduction in placental size (Fig. 4). At Day 10 of gestation, the TgE26 placenta was measured at 47% of a CD1 control placenta, whereas at Day 12, placental area was measured at 44% of a time-matched CBA/J placenta. Placentae of TgE26 were composed of giant cells, labyrinthine trophoblast, and spongiotrophoblast. The mesometrial triangle region was absent at all gestational days studied. Furthermore, GMG cells were essentially absent from the implantation sites. Between Days 10 and 17 of gestation, progressive histological changes occurred in the maternal arterioles of implantation sites containing normal fetuses (Fig. 5). At Day 10 of gestation, arteriole walls showed a thickening of the media and adventitia, and the vascular endothelium appeared cuboidal. By Day 12 of gestation, arteriole walls continued to thicken, and deposits of lipidlike material and foam cells were observed. The vascular endothelium now appeared flattened. One Day 12 litter containing 7 viable fetuses and resorbing fetus was serially sectioned. Every implantation site demonstrated the placental and vascular pathology described above. By Day 14 of gestation, the media and adventitia appeared thicker still, and the endothelium was discontinuous (Fig. 5, A-D). Leakage of blood had occurred into the surrounding decidua, and a neutrophil influx was present. The uterine vascular changes seen histologically in the TgE26 mice were restricted to the second half of pregnancy since blood vessels in virgin and postpartum uteri were normal (Fig. 5, E and F). At sites of resorbing TgE26 embryos, vascular thrombosis was observed, and a neutrophil influx appeared before Day 14 (Fig. 5G). Figure 5H shows a uterine arteriole from a CBA/J female at Day 12 of pregnancy. These changes were not found in major blood vessels (aorta, carotid) or organ vasculature (kidney, lung, liver) of pregnant or nonpregnant TgE26 mice (Fig. 6). Assessment of Maternal Versus Fetal Contributions TgE26 females were mated to CDI males to assess the role of an immune-deficient fetus in the pathology of the TgE26 females between Days 10 and 18 of gestation (Table 2). Implantation sites were studied between Days 8 and 18 of gestation. Viability in individual litters ranged from 50 to 100%, with a mean fetal viability of 87.4% (mean litter size: 8.7; n = 10 litters; 87 implantation sites). Histologically the implantation sites (n = 16) resembled those of TgE26 homozygous matings at Days 8 and 10 of gestation (Fig. 7A). At Day 8, implantation sites appeared normal. At Day 10, most placentae were small, there was no metrial gland or GMG cells, the walls of the arterioles were thickened, and the endothelium was raised. However, at Days 12-14 the placentae did not resemble those seen in TgE26 females carrying immune-deficient fetuses. The placenta was more disorganized, and there was considerable variation in appearance between implantation sites. Trophoblast giant cells had moved adjacent to the decidual arterioles, and the walls of these vessels appeared to have thinned. Unusual structures were seen in the labyrinthine region that included extremely large syncytia, with many peripherally located nuclei surrounding a vacuolated cytoplasm (Fig. 7C). In some specimens, extensive smooth muscle development was found adjacent to the labyrinthine trophoblast (Fig. 7F). Since animals heterozygous for the TgE26 transgene are not fully immune-competent [27], pregnancies having fully immune-competent CD 1 fetuses were created. Blastocysts were transferred to both uterine horns of five pseudopregnant TgE26 females. Of 49 CD1 embryos trans-
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3
i
3 wksof age-male
3 wks ofage female
7 wksof age -male
7wks of age -female
FIG. 2. Histogram comparing the weights of TgE26 and Tg2978 mice at 3 and 7 wk of age (p < 0.001). TgE26 mice weighed 29% (both males
and females) less than Tg2978 mice at 3 wk of age, and 27% less at 7 wk of age.
ferred, 42 implanted and were viable at Days 10-14 of gestation. Four of the TgE26 females received only CD1 embryos and carried litters of 8-10 embryos (8, 9, 10, 10 embryos). A single TgE26 female received CD1 embryos in one uterine horn and TgE26 embryos in the opposite horn. The former embryos implanted (n = 5) whereas the latter did not. Histologically, at Day 10, the immune competence of the fetus had no influence on the implantation site; that is, there was no development of metrial gland or GMG cells, and the media and adventitia of the decidual blood vessels were thick. At Days 12-14, the uterine histology was discordant to that seen in TgE26 mothers carrying an immune-deficient fetus. The position of the trophoblast giant cells was unusual. They appeared to form the walls of many but not all blood vessels (Fig. 7B). The remaining blood vessel walls were not thick. In many implantation sites, the decidual area contained very small blood vessels that appeared to represent the development of fine branching (Fig. 7E). In some implantation sites, the decidual area appeared to be filled with adipose tissue (Fig. 7D), similar to the region seen in the p561ck-ck-.IL-2R3-/ IL-2R3- mice (Fig. 1C).
FIG. 3. TgE26 mice exhibited a progressive loss of viable fetuses that was detected by Day 10 of gestation. This is abnormal when compared to random-bred CD1 mice or inbred CBA/J and C3H/HeJ mice.
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FIG. 4. Photomicrographs of pregnancies in TgE26 females carrying homozygous, immune-deficient TgE26 offspring (A-C, E) and in CD1 control mice (D, F). On Days 6 (A)and 8 (B)of gestation, implantation sites were assessed to be normal (arrow, conceptus). Days 10 (C)and 12 (E)and Day 14 implantation sites showed a small placental (p)size, the absence of metrial gland formation (-mg, C), and a relative acellularity in regions expected to be decidua, compared to controls (D) that displayed a metrial gland (mg) and a normal cellularity of decidua (d). The arrows in C and D demarcate the limit of trophoblast giant cells. Few PAS+ cells were present in the TgE26 mouse (E); an arrow points to a small, poorly granulated uNK cell. The typical size, granularity, and frequency of uNK cells (arrows) found in immunecompetent mice is shown in F.A-D) H&E; E,F) PAS. A, B) bar = 570 ~pm; C, D) bar = 1000 Vtm; E, F)bar = 50 p.m.
In the reverse experiment, six CD1 females received TgE26 blastocysts. Three of these also received CD1 blastocysts in the opposite uterine horn. All CD1 blastocysts implanted. Of 32 TgE26 blastocysts transferred, only 13 implanted. Two of the three recipients receiving TgE26 blastocysts that did not implant, successfully carried CD1 fetuses in the opposite uterine horn. Histologically, the TgE26 implantation sites in CD1 were normal, with metrial gland and GMG cell development (data not shown). DISCUSSION To understand the functions of GMG cells during normal pregnancy, a lineage depletion strategy was pursued. Previous antibody treatments using NKI.1 and asialo GM1 antibodies, the phenotype markers for mature GMG cells, were not successful in depleting GMG cells from pregnant animals [6]. Since GMG cells may have a major role at
implantation [28], we chose to treat females during the implantation period with Ly-49G2, a marker of uterine NK cells in the virgin and early-pregnant uterus. Ly-49G2 antibody treatment reduced uterine cell numbers in nonpregnant mice to 20-30% of the levels in controls, which is consistent with the maximal depletion achieved by Ly-49G2 in spleen by others [29]. However, Ly-49G2 treatment over the implantation period had no effect on pregnancy outcome. Further, the treatment did not reduce the frequency of Ly-49G2+-reactive or PAS-reactive GMG cells in pregnant animals assessed at either Day 7 or Day 12 of gestation. There are several possible explanations for these observations. First, residual Ly-49G2+ cells may be sufficient to maintain pregnancy. Second, it is possible that Ly-49G2+ cells were depleted but recovered quickly, by increased cell proliferation following cessation of antibody treatment. Third, depletion of cells may not in fact have
GESTATION IN NK CELL-DEFICIENT MICE
175 FIG. 5. Photomicrographs showing the histological changes in uterine arterioles of TgE26 females mated by TgE26 males between Days 8 and 14 of gestation, stained with H&E. At Day 8 of gestation, TgE26 vessel histology (A)was similar to that seen in control CBA mice (H). The common features included a continuous, flat endothelial cell layer (arrow). By Day 10 of gestation in TgE26 mice (B), the endothelial cells appeared cuboidal with a marked thickening of the arteriolar media and adventitia (asterisks). At Day 12 of TgE26 gestation (C), the endothelium appeared to have become flat (arrow), and the media remained thick. The deposition of lipid and the presence of foam cells (arrowheads) were apparent within the media and adventitia. By Day 14 of TgE26 gestation (D), the continued increase in vessel wall thickness (asterisks) was highly conspicuous. Loss of vessel integrity was observed in association with a discontinuous endothelium (large arrow) and led to blood leakage (arrowhead). Neutrophils were now present within blood vessels (small arrows). Arterioles found in virgin (E)and postpartum (F)TgE26 uteri were assessed as normal. Thus, changes in the uterine arterioles were restricted to midand late pregnancy. A large thrombus (G) occluding the vessel lumen was observed in association with a resorbing Day 12 TgE26 conceptus. Time-matched control CBA/J uterine arterioles were used for comparison. A Day 12 CBA/J artery is presented (H) as representative of endothelial height (arrow) and wall thickness (asterisks). A-D, G, H) bar = 50 m; E, F) bar = 20 Vm.
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FIG. 6. The endothelial changes and progressive thickening of the media and adventitia seen in the pregnant uteri of TgE26 females after Day 9 of gestation appear to be restricted to decidua associated arterioles, since these features were not observed in arterioles of any other organs or in the major blood vessel examined in nonpregnant (A, C, E)or pregnant (B, D, F) TgE26 mice. In the kidney (A,B), arrows show the typical height of the endothelium. The aorta (C,D) and carotid artery (E, F) are also depicted. A, B) bar = 50 m; C-F) bar = 10 rim; all H&E-stained.
been achieved, but rather down-regulation of Ly-49G2 surface antigen expression may have occurred on fully functional cells. Finally, Ly-49G2+ cells may not be the precursors of GMG cells. To increase the dose of antibodies used for depletion TABLE 2. In utero fetal viability of TgE26 females mated to CD1 males. Day of gestation 8 10 12 14 18
No. of females
No. of viable/ total implants
No. of corpora lutea
1 3 2 1 3
10/10 20/26 15/20 7/7 24/24
14 36 20 10 26
experiments would increase handling and the frequency of i.p. injections in pregnant animals. Since these stressors might also promote pregnancy failure, an alternative genetic depletion strategy was investigated. The preliminary survey indicated that expression of the IL-2 gene was not an important element for GMG cell differentiation since GMG cells were found with normal frequency, appearance, and localization in the IL-2-/- and IL-2-/-.3 2 m-/pregnancies. Two strains were identified that lacked GMG cells and metrial gland development, the p561 ck lick .IL2R3-/IL-2R3-
and TgE26. Because the separate p56ck
and IL-2RP3-/IL-2R3- mutations were not available for study, only TgE26 was investigated. From the studies of TgE26 pregnancies, the lineage of GMG cells has finally been established as NK. Although TgE26 mice are also T cell-deficient, the presence of GMG cells in the T cell-deficient nu/nu and scid/scid mice previously precluded GMG Ick
GESTATION IN NK CELL-DEFICIENT MICE
177 FIG. 7. Histological observations of implantation sites from TgE26 females pregnant with immune-competent fetuses (CD1 x TgE26, [A, C, F] or CD1 fetuses [B, D, El). No anomalies were noted at Days 6 or 8 of gestation. At Day 10 of gestation, implantation sites in both types of pregnancies resembled those with TgE26 fetuses; in particular, thick arterioles and cuboidal endothelium. A illustrates these features for a Day 10 F1 fetus (asterisk, thickened media and adventitia). The progression of gestations involving immune-competent fetuses beyond Day 10 was dissimilar to the gestations involving homozygous TgE26 immune-deficient fetuses. A variety of histopathology was seen that was inconsistent between fetuses. The histology may represent compensation that modified the vascular damage seen in TgE26 carrying immune-deficient fetuses. Trophoblast giant cells (arrowheads) were unusually located throughout the implantation sites, specifically, adjacent to the myometrium at the base of the placenta, intimately associated with decidual vessel walls, on Days 12 and 14 (B) of gestation. At Days 12 and 14 (C) of gestation, some placentae contained large synctitia, with peripheral nuclei and central vacuolated cytoplasm. In some Day 14 implants (D), the decidual site appeared to be filled with adipose tissue (ad) and was reminiscent of that seen in the p56' c k /Ik-.IL-2RP-.IL-2RP- implantation sites (m, myometrium). Some implantation sites containing CD1 fetuses revealed an increase in the number of small blood vessels present in the decidual region suggesting fine branching or collateral recruitment (E, Day 12). In some but not all implants, a significant increase in smooth muscle cell proliferation (arrows) was observed adjacent to the labyrinthine trophoblast (Ip) (F). Bar = 50 m.
cells from being T cells [2, 17]. We recommend that the term GMG cells be retired and replaced by use of the term uterine NK (uNK) cells. Further, significant new insights into the potential functions of uNK (GMG) cells have been provided. It is apparent that uNK cells are not essential for implantation because the numbers of implantation sites in TgE26 did not differ from those of controls. Onset of fetal loss was detected grossly at Day 10. No changes were observed in the ovaries accounting for this loss. However, the onset of fetal loss did coincide with the detection of histological changes in the placenta at Day 10. Thus, it appears that the important time for uNK cell function is postimplantation in association with placental differentiation and establishment of the placental circulation. Uterine NK cells do not appear to be involved in signaling events contributing to the differentiation of trophoblast cells, because all three layers of the trophoblast were
present in TgE26 and p56l ck aCk-.IL-2RP-/IL-2R3- placentae. The hypothesis that uNK cells limit trophoblast invasion is widely held and would predict trophoblast overinvasion in these strains. This was not observed. A consistent observation in TgE26 mice was small placental size throughout the second half of gestation. The consequences of this would be small fetuses and neonates [30]. TgE26 mice were smaller than control animals at both weaning and adulthood, suggesting that small placentae have a longterm influence on postnatal development as postulated in humans [31,32]. In the p561ck-/Ick-.IL-2RP-/IL-2R[ mice, the placental size appeared normal, but the Day 14 decidua did not. Since these mice lack uNK cells, caution must be exercised in interpretation of the findings in TgE26. To evaluate the role of insertional site mutagenesis in the TgE26 phenotype, studies are in progress to evaluate the reproductive performance of homozygous Tg2978 animals, which will be NK cell-deficient. Further, crosses to
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place the TgE26 transgene onto a different genetic background have been undertaken. The major histological abnormalities associated with the small TgE26 implantation sites were vascular. The lesions were progressive after they were first recognized and are suggestive of arteriosclerosis associated with hypertension [33]. The vessel wall changes indicative of arteriosclerosis associated with hypertension seemed to be localized to the uterus: vascular changes were not present in other vessels (aorta, carotid) and organs (lung, kidney, liver) in pregnant TgE26 mice or in the postpartum uterus. A leading cause of human perinatal mortality is pregnancy-induced hypertension and preeclampsia [34]. Diagnostic features of preeclampsia include elevated systemic blood pressure and proteinuria seen in the second half of gestation. Preliminary studies in TgE26 have found that mothers carrying immune-deficient TgE26 fetuses (n = 2) have high levels of proteinuria (> 500 mg/dL) late in gestation that fall to normal levels (< 30 mg/dL) by 24 h postpartum. The major histological finding in placentae from women with preeclampsia is reduced trophoblast invasion into the maternal spiral arteries. Endovascular trophoblast is not characteristic of the mouse placenta; however, the finding that trophoblast associates with the maternal blood vessels in TgE26 females carrying immune-competent fetuses suggests that trophoblast can respond to vascular changes in the pregnant mouse uterus. The movement of trophoblast in these pregnancies appeared to be an attempt to restore normal placental function since no fetal loss occurred in utero. An improvement in the ability of TgE26 females to carry fetuses beyond Day 10 of gestation when the fetus is immune-competent rather than immune-deficient suggests that the fetus itself influences placental vasculature. This would most likely be accomplished by fetal cytokines crossing the placenta to induce a maternal response. The major cytokine-producing environment found in these immune-competent fetuses that would be absent from homozygous TgE26 immune-deficient fetuses between Days 12 and 14 gestation would be the maturing thymus. The cytokine products of this environment are unlikely to be those produced by uNK cells in normal mice since placental morphology remained abnormal. However, a shift in the time of availability of a common cytokine could also promote fetal survival. An alternative explanation is that the CD1 genotype in both of the immune-sufficient fetuses underlies the observed fetal survival. The vascular problems identified in TgE26 females carrying immune-deficient fetuses could be sufficient to account for the death of some of the fetuses. It is known that a reduced blood supply to developing fetuses results in a hypoxic, toxic, or nutrient-depleted condition that can lead to intrauterine growth retardation and fetal mortality [35]. Because fetal growth demands accelerate dramatically subsequent to development of the placental circulation and are at their highest levels in the second half of gestation, a narrowing of the decidual arterioles, hypertension, and destruction of endothelium followed by blood loss through vascular leakage could lead to a nutrient-deficient environment. To avoid the loss of the total litter, a high maternal cost [36], it is possible that normal fetuses may be terminated to spare some of their litter mates. This would be consistent with the finding that entire TgE26 litters were never lost after the onset of the observed placental changes. One interpretation of this series of experiments is that uNK cells function to regulate smooth muscle cell proliferation during pregnancy and promote smooth muscle cell
relaxation. Interferon-y has been shown to inhibit smooth muscle cell proliferation by nitric oxide (NO), a major vasodilator that acts on vascular smooth muscle in many sites, including the uterus [37, 38]. A number of histologists have reported that murine and human uNK cells show a strong association with blood vessels. In mice and rats, uNK cells are observed to frequently pass through the adventitia and media [1]; however, the direction of the cell movement is unknown. In 1970, Moore et al. [39] suggested that uNK (GMG) cells may be involved in blood pressure regulation, since hypertensive rats in which an artificial deciduomata had been induced experienced a moderation in blood pressure at the time uNK cells appeared. The role of progesterone on blood vessels and uNK cell appearance was not addressed. The results of the present study do not constitute proof that the absence of uNK cells causes small placentae, intrauterine growth retardation, fetal death, and postnatal runting. Reconstitution of the uNK cell lineage, using adoptive transfer of normal progenitor cells, should clarify the importance of uNK cells during pregnancy. If reconstituting the uNK cell lineage restores placental size and reproductive performance, then a role in pregnancy success can be firmly assigned to uNK cells. These studies are necessary in establishing a vital role for uNK cells in placental growth and gestational success. ACKNOWLEDGMENTS We thank Dr. L.H. Mason (National Cancer Institute, MD) for his generous gift of the Ly-49G2 antibody; Drs. T.W. Mak and H. Suzuki (Amgen Institute, ON) for the p56lck /ck .IL-2Rf3-/IL-2RW--, and IL-2-/ - mice; Mr. Andrew Moore and Ms. Heather House (OMAFRA) for providing access to and valuable assistance with the Northern Exposure System; Drs. Janet Rossant, Dean Percy, and Shigeto Yamashiro for advice and helpful discussions; and Mrs. Betty-Anne McBey and Mrs. Barb Mitchell for technical assistance.
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