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 http://www.jimmunol.org/content/171/6/2937 Downloaded from References This article cites 42 articles, 12 of which you can access for free at: http://www.jimmunol.org/content/171/6/2937.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on October 7, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 (4–8). 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 (9–12). 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 (14–17). 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 Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00 2938 IL-15, IRF-1, AND IRF-2 IN uNK CELL DIFFERENTIATION 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 (18–21). 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; 30–35 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-15–5Ј,5Ј-GCCATAGCCAGCTCATCTTC-3Ј; IL-15–3Ј,5Ј- establish, because unsuccessful clinical outcomes in human preg- GCAATTCCAGGAGAAAGCAG-3Ј; IRF-2–5Ј,5Ј-ACACTGGAGGAA nancies are being attributed to imbalance in IL-15 (17). Mice lack- GAGGAGCA-3Ј; IRF-2–3Ј,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.