Pregnancy Differentiation and Function During IFN Regulatory

Assessment of Requirements for IL-15 and IFN Regulatory Factors in Uterine NK Cell Differentiation and Function During Pregnancy This information is current as of October 7, 2021. Ali A. Ashkar, Gordon P. Black, Qingxia Wei, Hong He, Luchuan Liang, Judith R. Head and B. Anne Croy J Immunol 2003; 171:2937-2944; ; doi: 10.4049/jimmunol.171.6.2937

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Assessment of Requirements for IL-15 and IFN Regulatory Factors in Uterine NK Cell Differentiation and Function During Pregnancy1

Ali A. Ashkar,2* Gordon P. Black,† Qingxia Wei,‡ Hong He,† Luchuan Liang,§ Judith R. Head,§ and B. Anne Croy†

In mouse and human, precursors of NK cell lineage home to decidualizing uteri. To assess the requirement for IL-15, an essential cytokine for NK differentiation in lymphoid tissue, on uterine NK (uNK) cell differentiation, implantation sites from IL-15؊/؊ mice were analyzed histologically. IL-15؊/؊ implantation sites had no uNK cells, no spiral-artery modification, and lacked the decidual integrity found in normal mice. IL-15؊/؊ recipients of C57BL/6 marrow displayed similar pathology. However, implantation sites ؊/؊␥ ؊/؊ ؊/؊ from recombination-activating gene-2 c (alymphoid) recipients of IL-15 marrow showed normal uNK cells, modified spiral arteries, and well-developed decidua basalis. Deletion of the IFN-regulatory factor (IRF)-1, but not IRF-2 (factors important Downloaded from in peripheral NK cell differentiation) limited but did not prevent uNK cell development. In situ hybridization localized IRF-1 ؊/؊ ؊/؊␥ ؊/؊ largely to placental trophoblast cells. IRF-1 marrow transplanted into recombination-activating gene-2 c displayed ,competence for full uNK cell differentiation. IL-15 mRNA expression at implantation sites of IRF-1؊/؊ and C57BL/6 was similar suggesting that, unlike in bone marrow and spleen, IRF-1 does not regulate IL-15 in the pregnant uterus. Terminal differentiation of uNK cells was not promoted in pregnant IRF-1؊/؊ mice by 5-day infusion of murine rIL-15, suggesting that IRF-1 deficiency rather than IL-15 deficiency limits uNK cell differentiation in these mice. Further, IRF-1 regulates placental growth, birth weight, http://www.jimmunol.org/ and postnatal growth of offspring. These studies indicate that uNK cell development and maturation share some aspects with NK cell development in other tissues, but also display distinctive tissue-specific regulation. The Journal of Immunology, 2003, 171: 2937–2944.

terine NK (uNK)3 cells, an NK cell subset, are the most arteries and veins as they enter and leave implantation sites (4). frequent lymphocytes found within implantation sites uNK cells are the major but not sole sources of IFN-␥ on the during the first half of normal pregnancy in rodents, mesometrial aspect of each implantation site (5). U Ϫ Ϫ ϩ Ϫ/Ϫ women, and nonhuman primates (1, 2). In mice, terminally differ- The absence of uNK cells in tg⑀26 (NK ,T ,B ), IL-2R␤ by guest on October 7, 2021 Ϫ Ϫ/Ϫ␥ Ϫ/Ϫ entiating uNK cells are first recognized at gestation day (gd) 5 (1, (NK ), and recombination-activating gene-2 (RAG-2) c 3) in the last region of each implant site to show transformation of (NKϪ,TϪ,BϪ) mice, or absence of IFN-␥ signaling in IFN-␥Ϫ/Ϫ, fibroblasts to decidual cells, the decidua basalis (DB). uNK cells IFN-␥R␣Ϫ/Ϫ, and Stat-1Ϫ/Ϫ mice during pregnancy is associated rapidly increase in number by proliferation in DB, and then in a with structural stability rather than modification of the branches of myometrial region called the mesometrial triangle. By gd 10 of the the uterine arteries (called decidual spiral arteries (SAs)) that sup- 19- to 20-day pregnancy, uNK cells form transient, pregnancy- ply the conceptuses. Normal, pregnancy-induced reduction of the associated mural structures at each implantation site called me- vascular smooth muscle coat, and arterial dilation and elongation sometrial lymphoid aggregates of pregnancy (MLAps) or metrial are restricted (4Ð8). In addition to triggering vascular changes, glands. The MLAps surround the branches of maternal uterine uNK cell-derived IFN-␥ promotes the terminal steps in uterine stromal cell transformation into decidua and controls uNK cell *Department of Pathology and Molecular Medicine, McMaster University, Hamilton, maturation (judged by diameter and cytoplasmic granularity) and Ontario, Canada; †Department of Biomedical Sciences, Ontario Veterinary College, senescence (7). Mechanisms by which uNK cell-derived IFN-␥ University of Guelph, Guelph, Ontario, Canada; ‡The Arthur and Sonia Labatt Brain Tumor Research Centre, The Hospital for Sick Children, Toronto, Ontario, Canada; exerts these actions are not defined. and ¤Department of Obstetrics and Gynecology, University of Texas Southwestern In lymphoid organs, IL-15 plays a key role in the maturation of Medical Center, Dallas, TX 75390 NK progenitor cells (9Ð12). The mouse uterus initiates transcrip- Received for publication April 30, 2003. Accepted for publication July 16, 2003. tion of IL-15 following the onset of decidualization until gd 11, The costs of publication of this article were defrayed in part by the payment of page when it is lost (13). IL-15 has been demonstrated in the human charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. uterus throughout the menstrual cycle. Human uterine IL-15 mes- 1 These studies were supported by Natural Sciences and Engineering Research Coun- sage and protein are elevated in the secretory phase of the men- cil; Ontario Ministry of Agriculture, Food, and Rural Affairs; National Institutes of strual cycle, when decidualization begins (2) and in early preg- Health (HD32552); and Ministry of Science, Research, and Technology of Iran. nancy when decidual tissue is greatly increased (14Ð17). Human 2 Address correspondence and reprint requests to Dr. Ali A. Ashkar, HSC-4H30G, uterine IL-15 expression is most prominent in perivascular cells Department of Pathology and Molecular Medicine, McMaster University, 1200 Main Street West, Hamilton, Ontario, Canada L8N 3Z5. E-mail address: ashkara@ surrounding the decidual SA in the late cycle and in the endothelial mcmaster.ca cells of these arteries during early pregnancy (15). In lymphoid 3 Abbreviations used in this paper: uNK, uterine NK; gd, gestation day; DB, decidua tissue, the transcription factors IFN-regulatory factor (IRF)-1 and basalis; MLAp, mesometrial lymphoid aggregate of pregnancy; RAG, recombination- activating gene; SA, spiral artery; IRF, IFN-regulatory factor; PAS, periodic acid- IRF-2 regulate NK cell differentiation. IRF-1 binds to promoter Schiff; BM, bone marrow; m, murine. sequences in the IL-15 gene, whereas IRF-2 appears to regulate the

IL-15R. IRF-1 and IRF-2 are key regulators of IL-12-induced Analysis of expression of IL-15 and IRF-2 mRNA by RT-PCR ␥ IFN- production by NK cells (18Ð21). However, uterine IRF-1 is Total RNA was isolated from freshly dissected tissue using RNeasy mini only abundantly expressed in lumenal and glandular epithelium kit (Qiagen, Hilden, Germany), according to the manufacturer’s instruc- and not uterine stroma in humans (22). This is the same site to tions. Two micrograms of total RNA were reverse transcribed using first- which IRF-2 has been localized in the sheep uterus, the only spe- strand cDNA synthesis kit (Amersham Pharmacia Biotech, Piscataway, cies in which uterine IRF-2 has been investigated (23). Thus, there NJ). cDNA were amplified using the following conditions: 94¡C for 6 min; 30Ð35 cycles of 94¡C for 45 s, 56¡C for 45 s, and 72¡C for 45 s; and an is circumstantial, but no direct evidence that IL-15 is important for extra 7 min at 72¡C following the last cycle. The PCR primers were as uNK cell differentiation during pregnancy. This is important to follows: IL-15Ð5Ј,5Ј-GCCATAGCCAGCTCATCTTC-3Ј; IL-15Ð3Ј,5Ј- establish, because unsuccessful clinical outcomes in human preg- GCAATTCCAGGAGAAAGCAG-3Ј; IRF-2Ð5Ј,5Ј-ACACTGGAGGAA nancies are being attributed to imbalance in IL-15 (17). Mice lack- GAGGAGCA-3Ј; IRF-2Ð3Ј,5Ј-CAACAACCACCAGGGAGAGT-3Ј; ␤-actin-5Ј,5Ј-GCTACAGCTTCACCACCACA-3Ј; and ␤-actin-3Ј, ing IL-15 (10), IRF-1, and IRF-2 (24) are reproductively compe- 5Ј-ACATCTGCTGGTAGGTGGAC-3Ј. tent but have profound reductions in numbers of peripheral NK cells. Implantation sites have not been assessed in these strains to Northern blot analysis characterize uNK cell differentiation or quantify SA modification. For IRF-1 Northern analysis, total RNA was isolated from freshly dissected The present study was undertaken to define the role of IL-15 in tissue using the RNeasy mini or midi kits (Qiagen) according to the man- supporting the differentiation of uNK cells within mouse implan- ufacturer’s instructions. Total RNA from B6 spleen cells, which had been tation sites and to address the requirements for IRF-1 and IRF-2 in activated with murine (m)rIFN-␥ (100 U/ml) and LPS (200 ng/ml) for 6 h ␮ uNK cell differentiation. was used as positive control. Ten to 20 g of total RNA were electropho- resed in 1% denaturing agarose-formaldehyde gels and then transferred onto Hybond nylon membranes (Amersham Life Sciences, Oakville, On- Downloaded from Materials and Methods tario, Canada) according to standard protocols. Following electroblotting, Mice the RNA was cross-linked by UV irradiation, and the nylon membranes were stored at 4¡C until further processing. The mouse IRF-1 (574-bp) C57BL/6J (B6) and IRF-1Ϫ/Ϫ mice were purchased from The Jackson Lab- probe was generated from clone no. 777393 supplied by GenomeSystems Ϫ/Ϫ Ϫ/Ϫ␥ Ϫ/Ϫ oratory (Bar Harbor, ME). Breeding pairs of IRF-2 and RAG-2 c (St. Louis, MO). Radioactive probes were prepared using a commercially were kindly provided by Drs. T. Mak (University of Toronto, Toronto, prepared Rediprime random-primer DNA-labeling mixture (Amersham In- Ontario, Canada) and J. Di Santo (Pasteur Institute, Paris, France). Breed- ternational, Buckinghamshire, U.K.). After dissolving the Rediprime mix- Ϫ Ϫ 32 ing pairs of IL-15 / mice were provided by Immunex (Amgen, Thousand ture and DNA, 50 ␮Ci of radioactive nucleotide [␣- P]dCTP (specific http://www.jimmunol.org/ Oaks, CA). All strains of knockout mice used in this study were of B6 activity, 3000 ␮Ci/mmol; Amersham Life Sciences) was added. The mix- background. B6 mice were housed under conventional husbandry. All ture was incubated for 10 min at 37¡C. Reactions were stopped by adding other strains were bred under monitored, barrier husbandry in the Univer- 5 ␮l of 0.2 M EDTA. The radioactive probe was purified by centrifuging sity of Guelph (Ontario Ministry of Agriculture, Food, and Rural Affairs 300 ␮l of TEN through a Quick Spin column (Bio-Rad Laboratories, Her- Isolation Unit (Guelph, Ontario, Canada)). Females were syngeneically cules, CA) containing 1 ml of Sephadex G-50 at 2000 rpm for 2 min. mated. At specific gestation day, counting the copulation plug as day 0, Membranes supporting the RNA samples were first prehybridized for 1 h ϫ timed-pregnant mice were euthanized by CO2 inhalation, followed by cer- at 42¡C in a solution containing 5 sodium chloride/sodium phosphate vical dislocation. Uterine horns were dissected and examined for fetal vi- EDTA, 0.5% SDS, 5ϫ Denhardt’s solution, salmon sperm DNA (40 ␮g/ ability (implant size and color). Only viable implantation sites were chosen ml), and 50% formamide. Then, the prehybridization buffer was replaced for further study. Some of the pregnant B6 and IRF-1Ϫ/Ϫ mice were per- by hybridization buffer containing IRF-1 or 7S probes. Following over- by guest on October 7, 2021 mitted to give birth, and their pups were weighed at birth (Ͻ12 h), and 21 night hybridization at 42¡C, membranes were washed twice with low-strin- and 49 days of age. gency solution containing 2ϫ SSC and 1% SDS at 42¡C for 15 min, followed by a high-stringency wash with 0.1ϫ SSC and 0.1% SDS at 50¡C Morphological and morphometrical analyses of implantation for 10 min. Blots were rinsed in 5ϫ sodium chloride/sodium phosphate sites EDTA, and the hybridization signal was detected by autoradiography, using Kodak (Rochester, NY) x-ray film. For IL-15, total RNA extracted by Implantation sites were fixed in Bouin’s fixative (Fisher Scientific, Whitby, the guanidinium isothiocyanate method (25) was analyzed by Northern blot Ontario, Canada) overnight and in 70% ethanol until processed. All fixed hybridization as described, including stringent posthybridization washes tissues were processed into paraffin using standard methodology. Two or (26). Each lane contained 10 ␮g of total RNA. [32P]cRNA probes were three paraffin-embedded implantation sites from each pregnant mouse (four generated from a ribovector containing mIL-15 cDNA, generously sup- to five pregnant females per study group) were serially sectioned (7 ␮m) plied by A. Troutt (Immunex, Seattle, WA). and stained with H&E or periodic acid-Schiff (PAS), which stains the gly- coprotein in uNK cell granules. All stained slides were examined by light In situ hybridization for IRF-1 microscopy. The center section of each serially sectioned implantation site In situ hybridization was performed on paraffin-embedded sections of non- was identified, and for each implantation site, five median tissue sections pregnant gestation day-timed uteri from B6 and alymphoid mice. Clone no. on each side of the central section were also analyzed (n ϭ 11). Sections 777393 (supplied by GenomeSystems) was used for the mouse IRF-1 (574- were at least 42 ␮m apart to avoid duplicate counting of individual uNK bp) probe. Digoxigenin-labeled IRF-1 sense and antisense probes were cells. The number of uNK cells (PAS positive) per square millimeter in the made using Northern starter kit, according to the manufacturer’s instruc- MLAp and in the DB was counted at ϫ500 magnification using an eye- tions (Roche, Montreal, Quebec, Canada). In situ hybridization on 8-␮m piece micrometer grid of 1 mm2. The circular smooth muscle was used as sections was performed according to the nonradioactive in situ hybridiza- the dividing line between the MLAp and DB. Cross-sectional area mea- tion application manual (Roche). For the visualization of the RNA probe, surements of the MLAp, decidua, and placenta, and ratios for vessel:lumen the sections were incubated with 5-bromo-4-chloro-3-indolyl phosphate/ diameter line morphometry of the main decidual arteries, cut in cross- nitroblue tetrazolium liquid substrate system (Sigma-Aldrich, Oakville, section, were measured on the same slides that were used for uNK cell Ontario, Canada) for up to 24 h. numeration, using OPTIMAS image analysis software, version 6.2 (Opti- mas, Bothwell, MA). Treatment of pregnant IRF-1Ϫ/Ϫ mice with mrIL-15 Bone marrow (BM) transplantation IRF-1Ϫ/Ϫ mice received 500 ng of mrIL-15 (Research Diagnostics, Ϫ/Ϫ␥ Ϫ/Ϫ Ϫ/Ϫ Flanders, NJ) i.p., daily for 5 days, from gd 5. On gd 10, pregnant uteri Six- to 8-wk-old RAG-2 c or IL-15 females were used as re- were dissected and processed for analysis. The dosage and route of cipients of BM. Each recipient was pretreated with a single i.p. injection of mrIL-15 were selected based on previous in vivo experiments (10, 27). 5-fluorouracil (150 mg/kg) 48 h before BM infusion. Six- to 8-wk-old Ϫ/Ϫ Ϫ/Ϫ IL-15 , IRF-1 , or B6 mice were used as BM donors. BM cells were Statistical analyses pooled from femurs and humeri. Donor cells were depleted of RBC using lysis buffer. A total of 2 ϫ 107 viable BM cells was given i.v. to each The means of experimental groups were compared using two-factor 5-fluorouracil-treated female. Three weeks later, recipients were paired ANOVA. The between-mice and the between-implantation sites variances Ϫ/Ϫ␥ Ϫ/Ϫ Ͻ with RAG-2 c males for mating and euthanized at gd 12. were considered as analytic factors ( p 0.05). Groups with significantly The Journal of Immunology 2939 Downloaded from

FIGURE 1. Implantation site anomalies in the absence of uNK cells. Photomicrographs of implantation sites from gd 12 mice. IL-15Ϫ/Ϫ mice (a and Ϫ/Ϫ␥ Ϫ/Ϫ a2) and RAG-2 c (b and b2) are genetically devoid of uNK cells. MLAp structure does not develop in the myometrial wall of these mice. The implantation site in the normal C57BL/6 congenic controls shows development of uNK cells, as indicated by arrows, and MLAp (c and c2). Implantation sites of mice lacking uNK cells possess unmodified decidual spiral arteries, characterized by a thicker blood vessel wall (a1 and b1, compared with c1) and higher wall:lumen ratios. The DB is also hypocellular (a and b) compared with the C57BL/6 control (c). The uNK cell-positive C57BL/6 mouse has http://www.jimmunol.org/ modified SAs, showing a much thinner blood vessel wall (c1). SAs in the C57BL/6 control are considered to be modified, whereas those arteries in mice without uNK cells retain the appearance of those in a virgin uterus. Arterial modification in C57BL/6 occurs between gd 9.5 and 10.0. Sections were stained with PAS. M, Myometrium. a–c, ϫ25; a1, a2, b1, b2, c1, and c2, ϫ400. by guest on October 7, 2021

FIGURE 2. IL-15 is the key molecule for uNK cell differentiation and maturation. Photomicrographs of gd 12 implantation sites in BM transfer Ϫ/Ϫ␥ Ϫ/Ϫ Ϫ/Ϫ recipients. a, In RAG-2 c recipients of IL-15 marrow, fully differentiated uNK cells are found in abundance (a1), and SA dilation occurs (a2). b, In IL-15Ϫ/Ϫ recipients of C57BL/6 marrow, no uNK cell differentiation occurs (b1), and no modiﬁcation of recipient arteries occurs (b2). c,In Ϫ/Ϫ␥ Ϫ/Ϫ RAG-2 c recipients of C57BL/6 marrow, uNK cell differentiation (c1) and SA modiﬁcation (c2) occur. Precursor cells receiving IL-15 signals differentiate into uNK cells. The MLAp of uNK containing implantation sites is well developed. d, IL-15 mRNA was detected by RT-PCR at the Ϫ/Ϫ␥ Ϫ/Ϫ implantation sites of RAG-2 c mice. Lane 1, DNA ladder; lane 2, nonpregnant uterus; lane 3, DB; and lane 4, placenta from gd 10. Sections were stained with PAS. a–c, ϫ25; a1, a2, b1, b2, c1, and c2, ϫ400. 2940 IL-15, IRF-1, AND IRF-2 IN uNK CELL DIFFERENTIATION

FIGURE 3. uNK cells contribute to SA modiﬁca- tion. Histograms are presented to summarize the mor- phometric analyses. a, The complete absence of uNK Ϫ/Ϫ␥ Ϫ/Ϫ Ϫ/Ϫ cells in RAG-2 c mice, IL-15 mice, and IL-15Ϫ/Ϫ recipients of C57BL/6 BM is indicated. In contrast, large numbers of uNK cells are present in Ϫ/Ϫ␥ Ϫ/Ϫ Ϫ/Ϫ RAG-2 c recipients of C57BL/6 or IL-15 BM, and these are equivalent to numbers in B6 implantation sites. Absence of uNK cells is shown by x. b, Vessel-to-lumen diameter measurements are presented. These ratios were similar at gd 12 in the strains without Ϫ/Ϫ␥ Ϫ/Ϫ Ϫ/Ϫ uNK cells (RAG-2 c , IL-15 , C57BL/6 BM- engrafted IL-15Ϫ/Ϫ). Lower ratios, indicative of wall thinning and lumen diameter increase, were induced by cells derived from IL-15Ϫ/Ϫ or C57BL/6 BM grafts in Ϫ/Ϫ␥ Ϫ/Ϫ RAG-2 c mice. Data were compiled from 11 measurements per implantation site (four to ﬁve

mothers per group; two to three implantation sites Downloaded from per mother). http://www.jimmunol.org/

FIGURE 4. IRF-1, but not IRF-2, is important for completion of uNK cell development. Photomicro- graphs of implantation sites of gd 12. a, IRF-1Ϫ/Ϫ mice have some uNK cells that appear poorly differentiated due to their small diameter and cytoplasmic hypogranularity (a2), unmodiﬁed SAs (a1), hypocel-

lular DB, and an underdeveloped MLAp (a). b, IRF- by guest on October 7, 2021 2Ϫ/Ϫ mice appear similar to genetically normal mice. They differentiate large numbers of uNK cells (b2), undergo SA modiﬁcation (b1), and develop a normal MLAp. c, Northern analysis for IRF-1 mRNA shows no expression on the mesometrial side of implantation sites from alymphoid and C57BL/6 mice. Lane 1,gd 6 mesometrial triangle RNA; lanes 2 and 3,gd10 MLAp RNA from B6; lanes 4 and 5, gd 6 and 10 RNA Ϫ/Ϫ␥ Ϫ/Ϫ from RAG-2 c mice, respectively; lanes 6 and 7, liver; and lane 8, spleen cells treated with IFN-␥ andLPSfor6haspositive control. d, RT-PCR for IRF-2 mRNA. Lanes 2–6 are from B6 mice, and lanes Ϫ/Ϫ␥ Ϫ/Ϫ 7–10 are from RAG-2 c mice. M, DNA ladder; lane 1, negative control (PCR of RNA without reverse transcription); lane 2, nonpregnant uterus; lane 3,gd 6; lanes 4–6, gd 10 MLAp, DB, and placenta, respectively; lane 7, nonpregnant uterus; lane 8,gd6;lanes 9 and 10, gd 10 DB and placenta, respectively. e1,In situ hybridization for IRF-1 mRNA in C57BL/6 mice. In the nonpregnant uterus, IRF-1 is found in uterine and glandular epithelial cells (e, as indicated by arrows, and e1). At gd 8, very strong hybridization was found on trophoblast giant cells (j and k, arrows, show positive cells). g, At gd 10, giant cells remained heavily labeled. f, Other placental cells were also positive, as were uterine epithelial cells adjacent to and at the antimesometrial aspect of the implantation sites. h, MLAp was negative, but signal was observed in dispersed decidual cells and SA walls. l, Sense probe negative control. a, a1, a2, b, b1, and b2, Sections were stained with PAS. a and b, ϫ25; a1, a2, b1, and b2, ϫ400. The Journal of Immunology 2941 different means were identiﬁed using Tukey’s test ( p Ͻ 0.05). Pup weights were compared by paired t test. Results Assessment of implantation sites in IL-15Ϫ/Ϫ mice IL-15 is essential for NK cell development in lymphoid tissue. Implantation sites of IL-15Ϫ/Ϫ mice differed from normal mice in that they completely lacked uNK cells. In addition, IL-15Ϫ/Ϫ mice did not develop MLAp, had exceedingly hypocellular DB, and failed to modify SA (Fig. 1, a vs c). The latter features resemble those seen in alymphoid mice (Fig. 1b) but are more pronounced. Placental cross-sectional areas in IL-15Ϫ/Ϫ mice were similar to those in time-matched normal controls (data not shown), and no elevated fetal loss (fetal loss, 2.3%; mean litter size, 8.2) was observed.

IL-15 is the key cytokine for uNK cell development To determine whether IL-15 was the sole limitation to uNK cell differentiation in IL-15Ϫ/Ϫ mice, IL-15Ϫ/Ϫ BM was transplanted into alymphoid mice, and B6 BM was transplanted into IL-15Ϫ/Ϫ Downloaded from or alymphoid recipients. Transplanted females were mated to non- transplanted, syngeneic males, and gd 12 implantation sites were studied. uNK cells differentiated from IL-15Ϫ/Ϫ BM and B6 BM in alymphoid mice to numbers equivalent to those found in normal mice. An MLAp developed, decidua became highly cellular, and arterial change was induced. However, transplanted B6 BM failed http://www.jimmunol.org/ to give rise to uNK cells in pregnant IL-15Ϫ/Ϫ hosts, no MLAp was present, decidual hypocellularity persisted, and arterial change was not induced (Figs. 2 and 3). Implantation sites of alymphoid mice were positive for IL-15 mRNA (Fig. 2d)

Assessment of IRFs in uNK cell development Because IRF-1 is thought to be essential for IL-15 expression in BM (Ogasawara et al., 1998), and both IRF-1 and -2 are important by guest on October 7, 2021 for NK cell development in marrow, implantation sites of IRF- 1Ϫ/Ϫ and IRF-2Ϫ/Ϫ mice were studied. They had no elevated fetal loss (fetal loss, 4.63%) compared with their congenic control. IRF- 2Ϫ/Ϫ sites did not differ histologically from implantation sites in normal mice (Fig. 4b), although RT-PCR showed that IRF-2 was FIGURE 5. Absence of IRF-1 leads to smaller placenta and lower pup abundantly expressed in virgin and pregnant uteri of B6 and alym- weight. Photomicrographs of implantation sites from gd 12 and 14 mice. a Ϫ/Ϫ phoid mice (d). In contrast, in IRF-1 mice after gd 6, implan- and b, In the IRF-1Ϫ/Ϫ mice, placental growth is impaired compared with tation sites were smaller grossly and in histologic cross-sectional age-matched C57BL/6 controls (c and d). e, IRF-1Ϫ/Ϫ placental cross- area than in time-matched B6 controls (Figs. 4a and 5). uNK cells sectional area is significantly smaller than that of age-matched controls. f Ϫ Ϫ differentiated in IRF-1 / mice but were few in number, small, and g, Weights of neonatal (Ͻ12 h), prepubescent (21 days), and adult (49 and hypogranular. In addition, MLAp regions were poorly devel- days) IRF-1Ϫ/Ϫ mice are significantly less than those of age-matched oped, and SA failed to modify (Fig. 4a). C57BL/6 controls. Sections were stained with PAS. a–d, ϫ20. P, Placenta. To assess whether small placental size affected fetal and/or postnatal development, IRF-1Ϫ/Ϫ litters were weighed at birth, wean- ing (21 days), and adulthood (49 days). Neonates were signifi- also positive, as were the uterine epithelial cells adjacent to and at cantly smaller than barrier husbandry-reared, age-matched B6, and the antimesometrial aspect of the implantation sites (Fig. 4f). The weight deficits persisted into adulthood (Fig. 5). uNK cell-rich MLAp was negative, but signal was observed in Microarray (GEM; Incyte, St. Louis, MO) data, based upon dispersed decidual cells and SA walls (Fig. 4h).

RNA collected from mesometrial triangles of B6 mice on gd 6 and Ϫ/Ϫ MLAps on gd 10, showed no IRF-1 expression (our unpublished IRF-1 precursors are competent to complete uNK cell observations). This was conﬁrmed by Northern analyses conducted differentiation on microdissected mesometrial triangle (gd 6) and MLAp (gd 10) To determine whether IRF-1Ϫ/Ϫ uNK precursors are competent for regions from B6 and alymphoid mice (Fig. 4c). To localize the normal maturation and numerical expansion in an IRF-1ϩ/ϩ uter- microdomains of IRF-1 expression within implantation sites, in ine environment, alymphoid mice were engrafted with BM from situ hybridization was conducted on virgin and pregnant uteri from IRF-1Ϫ/Ϫ mice. This established terminally differentiated uNK B6 and alymphoid mice. In both strains, signals in virgin uteri cells in recipients’ implantation sites (Fig. 6a; compare with c for were localized to the lumenal and glandular epithelia (Fig. 4, e and normal control, d for nonengrafted host, and f for donor implan- e1). At gd 8, in both strains, very strong hybridization was found tation sites). Reconstitution of alymphoid mice by IRF-1Ϫ/Ϫ BM in trophoblast giant cells (Fig. 4, j and k). At gd 10, giant cells induced MLAp development and reversed the anomalies in the remained heavily labeled (Fig. 4g). The now mature placenta was decidua and its arteries seen in implantation sites of untreated 2942 IL-15, IRF-1, AND IRF-2 IN uNK CELL DIFFERENTIATION

FIGURE 6. BM from IRF-1Ϫ/Ϫ mice is competent to restore uNK cells in an IRF-1-positive environment. Gestation day 12 implantation site photomicrographs. Ϫ/Ϫ␥ Ϫ/Ϫ RAG-2 c mice possess no uNK cells (d) and have unmodified SAs (e). Transferring IRF-1Ϫ/Ϫ BM Ϫ/Ϫ␥ Ϫ/Ϫ into RAG-2 c mice restores uNK cell proliferation and differentiation (a) compared with BM from B6 (c) and from IRF-1Ϫ/Ϫ (f). SAs of IRF-1Ϫ/Ϫ BM recipient mice are modified compared with unmanipu- Ϫ/Ϫ␥ Ϫ/Ϫ Ϫ/Ϫ lated RAG-2 c (b and e). g, IRF-1 mice are significantly deficient of uNK cells. Transplantation of Ϫ/Ϫ Ϫ/Ϫ␥ Ϫ/Ϫ IRF-1 marrow into RAG-2 c mice restores uNK cell frequency to control values. Sections were stained with PAS and magnified ϫ200. Downloaded from http://www.jimmunol.org/ hosts (Fig. 6, compare b with e). Quantification showed that not induce changes to IRF-1Ϫ/Ϫ implantation sites. The uNK cell IRF-1Ϫ/Ϫ BM reconstituted uNK cell numbers to those ob- population remained immature and was not increased in number. served in B6 (Fig. 6g). MLAp was not induced, and no significant changes were induced in SA or placental size from mrIL-15 treatment of IRF-1Ϫ/Ϫ mice IL-15 expression in the pregnant mouse uterus compared with untreated controls (Table I). Because BM cells from IL-15Ϫ/Ϫ and IRF-1Ϫ/Ϫ mice differentiate into uNK cells in alymphoid mice, presence of IL-15 in the recipient uteri was anticipated. Uterine mRNA from virgin and pregnant Discussion Ϫ/Ϫ alymphoid, IRF-1 and B6 mice was assessed for IL-15. RT- These studies clearly demonstrate that IL-15 is absolutely essential by guest on October 7, 2021 PCR, Northern (Fig. 7), and RNase protection assays (not shown) for the support of NK cell differentiation in the decidualizing all indicated that mRNA for IL-15 was induced after uterine de- uterus. Analyses of implantation sites in IL-15Ϫ/Ϫ mice revealed a cidualization in the three strains. Attempts to address IL-15 at the complete absence of uNK cells as well as pathology consistent protein level were unsuccessful. with that seen in other strains severely or totally deficient in uNK cells. The features included unmodified SA structure, poor devel- rIL-15 fails to restore normal uNK cell maturation and function Ϫ/Ϫ opment of decidua, and absence of MLAp development within the in IRF-1 mice uterine wall. These features do not compromise fetal viability or To further address whether anomalies at the implantation sites of postnatal survival under microbiological barrier husbandry and are IRF-1Ϫ/Ϫ mice were due to IL-15 protein deficiency, mrIL-15 was assumed to negatively reflect the major functions of uNK cells. No administered for 5 days to pregnant IRF-1Ϫ/Ϫ mice. Treatment did measures of fetal stress or postnatal physiology were performed.

FIGURE 7. IL-15 mRNA is found at implantation sites of C57BL/6 and IRF-1Ϫ/Ϫ mice. Total RNA was isolated from implantation sites on different days of gestation and from other tissues and analyzed for IL-15 mRNA expression. a, IL-15 mRNA was detected in nonpregnant uterus (gd Ϫ1) from all three strains of mice. The IL-15 message was weaker in IRF-1Ϫ/Ϫ than B6 spleen. SP; spleen; PL; placenta; control indicates RNA with no reverse transcription. b, Upper panel, Northern blot for IL-15 mRNA from implantation sites of IRF-1Ϫ/Ϫ and B6 mice at gd 8 (D8) and 12 (D12) (B6 mice had the same pattern of mRNA expression seen in IRF-1Ϫ/Ϫ mice). IL-15 mRNA in DB and placenta were much lower at gd 12 compared with gd 10. M, MLAp; D, DB; P, placenta. b, Lower panel, An ethidium bromide-stained gel is shown. The Journal of Immunology 2943

Table I. mrIL-15 failed to restore implantation site anomalies in IRF-1Ϫ/Ϫ mice

No. of uNK Cells Ϯ SEM Vessel:Lumen Ratios Placental Cross-Sectional MLAp DB of SA Ϯ SEM Area (mm2) Ϯ SEM

IRF-1Ϫ/Ϫ ϩ mrIL-15 17.4 Ϯ 3.57a 19.4 Ϯ 4.01a 2.19 Ϯ 0.22a 9.6 Ϯ 1.70a IRF-1Ϫ/Ϫ ϩ PBS 16.3 Ϯ 3.72b 20.1 Ϯ 4.13b 2.08 Ϯ 0.24b 9.0 Ϯ 1.58b B6 control 64.6 Ϯ 7.21 38.7 Ϯ 4.63 1.19 Ϯ 0.19 18.7 Ϯ 2.04

a No significant differences when compared with those of IRF-1Ϫ/Ϫ mice (PBS control), but significantly different compared with B6 control, p Ͻ 0.006. b No significant differences when compared with those of IRF-1Ϫ/Ϫ mice treated with mrIL-15, but significantly different compared with B6 control, p Ͻ 0.005.

The time course for transcription of mouse uterine IL-15 ex- cell differentiation steps. Vascular addressin VCAM could be a pression (13) mirrors that for development of uNK cells. Rare cells key IRF-1-regulated recruitment molecule (38). VCAM has unique are first found at gd 5 with significant numbers on gd 6 when IL-15 expression in DB and is postulated to be the major addressin hom- message is first reported. uNK cells proliferate within mouse ing NK cells to the uterus (31, 37). IRF-1 also contributes to vas- uterus from gd 6 to midgestation, achieve relatively stable peak cular remodeling events where it is antagonized by IRF-2 (39, 40). numbers between gd 10 and 12, and then gradually decline in IRF-2 appears to be an enhancer of VCAM-1 expression in myo- numbers (3, 28). IL-15 message in decidua is found to gd 11 and genic events outside of the vasculature (41). Downloaded from is then absent to term (13). Withdrawal of IL-15 would be the Endocrine control of IRF-1 and IL-15 gene expression has been simplest explanation for the disappearance of uNK cells. IL-15 proposed in human uterus and may account for the absence of withdrawal is likely gradual due to transendosomal cycling pro- IRF-1 regulation of murine uterine IL-15. Prolactin, a pituitary viding persistent surface-bound IL-15 in decidual stroma (29). Just hormone, is thought to enhance IRF-1 expression (42), whereas before parturition, ϳ10% of the peak uNK cell number remains. progesterone and PG, derived from ovary and placenta, are pro- These cells are shed with the placenta (28). Induction of IL-15 posed as regulators of IL-15 expression (14Ð16). However, these http://www.jimmunol.org/ transcription in SA endothelium, as seen in women, may also oc- mechanisms fail to explain regulation of NK cell differentiation in cur in mice. This could account for the highly localized appearance decidua, because the hormone effects are present systemically. of intravascular uNK cells within the component of the SA cours- Thus, additional unique regulatory mechanisms must be involved ing through the decidua (1, 30, 31) and for uNK cell absence from in IL-15 expression in decidualizing uterus. more superficial segments of the same vessels within the MLAp The results of this study suggest that uNK cell development and (32). This positional relationship may be of major functional im- maturation have aspects similar to NK cell development in other portance in elongation and dilation of SA, because both mouse and tissues but, in addition, have distinct differences. The absence of human uNK cells express endothelial cell mitogens and other va- IL-15 leads to the absence of both uNK and NK cells, whereas by guest on October 7, 2021 soactive molecules such as inducible NO synthase (33, 34) absence of IRF-1 results in partial deficiency of uNK and complete If IL-15 is the only growth factor essential for uNK cell differ- deficiency of NK cells. In the absence of IRF-2, uNK cells are entiation, as suggested by our marrow transplants, IL-15 regulation normal in number and function, but there is a severe deficiency of within uterus becomes an important question. In marrow, IRF-1 peripheral NK cells. This may suggest tissue-specific regulation of appears to be a key transcriptional activation factor for IL-15 (20). IL-15 in uterine stromal cells. Unique, IRF-1-regulated decidual Implantation sites in IRF-1Ϫ/Ϫ mice differ from those in any other cytokines may account for the skewing of NK cells features seen mouse strain reported to date. The uNK cell lineage was estab- in uterine compared with peripheral sites. lished, but it failed to fully differentiate numerically or morpho- logically. Furthermore, a major role for IRF-1 in trophoblast, par- Acknowledgments ticularly giant-cell, biology was defined. Over a period of 3 years, We thank Immunex, Inc., for providing the IL-15 cDNA probe and per- Ϫ Ϫ no drift was seen in the IRF-1 / phenotype of small placentae mission to receive breeding pairs of IL-15Ϫ/Ϫ mice from Dr. M. Caligiuri and small size of offspring. Small placentae may be the primary (Ohio State University, School of Medicine, Columbus, OH); Dr. T. W. Ϫ Ϫ Ϫ Ϫ cause of the intrauterine growth retardation, or there may be ad- Mak for providing IRF-1 / and breeding pairs of IRF-2 / mice; Lorena ditional primary deficits in fetuses as themselves during develop- Montoya, Martha Brackin, and the Ontario Ministry of Agriculture, Food, ment. 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