A chemokine,interferon (IFN)-gamma-inducible protein 10 kDa,is stimulated by IFN-tau and recruits immune cells in the ovine endometrium

Proper distribution of immune cells in the uterus is a prerequisite for successful implantation and subsequent placentation, but biochemical signals that govern such events have not been well characterized. In the present study, the cDNA of a chemokine, interferon (IFN)-gamma-inducible protein 10 kDa (IP-10), was identified from a cDNA subtraction study between uterine endometrial tissues from Day 17 pregnant and Day 15 cyclic ewes. The effect of IFN-tau on IP-10 expression and the involvement of IP-10 in the recruitment of immune cells were then investigated. Northern blot analysis revealed that large amounts of IP-10 mRNA were present during conceptus attachment to maternal endometrium and early placentation. IP-10 mRNA was localized to monocytes distributed in the subepithelial stroma of pregnant but not cyclic uteri. This finding was supported by the discovery of IP-10 mRNA expression in monocytes but not in lymphocytes, uterine epithelial cells, or stromal cells. Moreover, the expression of IP-10 mRNA by the monocytes was stimulated by IFN-alpha, IFN-gamma, and IFN-tau in a dose-dependent manner, but the expression of IP-10 mRNA by the endometrial explants was most stimulated by IFN-tau. In a chemotaxis assay, migration of peripheral blood mononuclear cells was stimulated by the addition of IFN-tau stimulated-endometrial culture medium, and the effect was significantly reduced by neutralization with an anti-IP-10 antibody. These results suggest that endometrial IP-10 regulated by conceptus IFN-tau regulates recruitment and/or distribution of immune cells seen in the early pregnant uterus.     

Regulation of conceptus adhesion by endometrial CXC chemokines during the implantation period in sheep

To gain a better understanding of biochemical mechanisms of conceptus adhesion to the maternal endometrium in ruminant ungulates, the present study was performed to clarify roles of chemokines and extracellular matrix (ECM) components in the regulation of ovine blastocyst attachment to the endometrium. In addition to the chemokine, interferon-gamma inducible protein 10 kDa (IP-10, CXCL10), the chemokine receptor, CXCR3, also recognizes two other chemokines; monokine induced by IFN-gamma (MIG, CXCL9) and IFN-inducible T cell alpha chemoattractant (I-TAC, CXCL11). Similar to CXCL10, CXCL9, and CXCL11 were expressed in the uterus during the peri-implantation period, and CXCL9 mRNA expression was stimulated in endometrial explants from day 14 cyclic ewes by the addition of IFN-tau or IFN-gamma. Without ECM components, conceptus cell adhesion was low on day 14 of gestation and exhibited a 2.5-fold increase on day 17; adhesiveness on day 20 was 1/10 of that on day 14. Among various ECM components examined, trophoblast adhesion was greatest when fibronectin was used. Although day 14 conceptuses did not show much adhesive activity to fibronectin, day 17 trophoblast, and day 20 chorionic membrane exhibited 2.3-fold and 50-fold increase, respectively, which was enhanced by treatment with CXCL9 or CXCL10. These results indicate that through endometrial fibronectin and chemokines, ovine conceptus cells gain the ability to attach to the endometrium during pre-implantation period; however, elucidation of molecular mechanisms by which the conceptus acquires the adhesive ability during this time period awaits further investigation.     

Alpha5beta1, alphaVbeta3 and the platelet-associated integrin alphaIIbbeta3 coordinately regulate adhesion and migration of differentiating mouse trophoblast cells

During blastocyst implantation, interaction between integrins on the apical surface of the trophoblast and extracellular matrix (ECM) in the endometrium anchors the embryo to the uterine wall. Strong adhesion of the blastocyst to fibronectin (FN) requires integrin signaling initiated by exogenous fibronectin. However, it is not known how integrin signaling enhances blastocyst adhesion. We present new evidence that the integrin, alphaIIbbeta3, plays a key role in trophoblast adhesion to fibronectin during mouse peri-implantation development. Trafficking of alphaIIb to the apical surface of the trophoblast increased dramatically after blastocysts were exposed to fibronectin, whereas other fibronectin-binding integrins, alpha5beta1 and alphaVbeta3, were resident at the apical surface before ligand exposure. Functional comparisons among the three integrins revealed that ligation of alpha5beta1 most efficiently strengthened blastocyst fibronectin-binding activity, while subsequent trophoblast cell migration was dependent primarily on the beta3-class integrins. In vivo, alphaIIb was highly expressed by invasive trophoblast cells in the ectoplacental cone and trophoblast giant cells of the parietal yolk sac. These data demonstrate that trafficking of alphaIIb regulates adhesion between trophoblast cells and fibronectin as invasion of the endometrium commences.     

Gene expression and production of the monokine induced by IFN-gamma (MIG), IFN-inducible T cell alpha chemoattractant (I-TAC), and IFN-gamma-inducible protein-10 (IP-10) chemokines by human neutrophils

Monokine induced by IFN-gamma (MIG), IFN-inducible T cell alpha chemoattractant (I-TAC), and IFN-gamma-inducible protein of 10 kDa (IP-10) are related members of the CXC chemokine subfamily that bind to a common receptor, CXCR3, and that are produced by different cell types in response to IFN-gamma. We have recently reported that human polymorphonuclear neutrophils (PMN) have the capacity to release IP-10. Herein, we show that PMN also have the ability to produce MIG and to express I-TAC mRNA in response to IFN-gamma in combination with either TNF-alpha or LPS. While IFN-gamma, alone or in association with agonists such as fMLP, IL-8, granulocyte (G)-CSF and granulocyte-macrophage (GM)-CSF, failed to influence MIG, IP-10, and I-TAC gene expression, IFN-alpha, in combination with TNF-alpha, LPS, or IL-1beta, resulted in a considerable induction of IP-10 release by neutrophils. Furthermore, IL-10 and IL-4 significantly suppressed the expression of MIG, IP-10, and I-TAC mRNA and the extracellular production of MIG and IP-10 in neutrophils stimulated with IFN-gamma plus either LPS or TNF-alpha. Finally, supernatants harvested from stimulated PMN induced migration and rapid integrin-dependent adhesion of CXCR3-expressing lymphocytes; these activities were significantly reduced by neutralizing anti-MIG and anti-IP-10 Abs, suggesting that they were mediated by MIG and IP-10 present in the supernatants. Since MIG, IP-10, and I-TAC are potent chemoattractants for NK cells and Th1 lymphocytes, the ability of neutrophils to produce these chemokines might contribute not only to the progression and evolution of the inflammatory response, but also to the regulation of the immune response.     

Fibroblast growth factor-10: a stromal mediator of epithelial function in the ovine uterus

Fibroblast growth factor-10 (FGF-10) is a stromal-derived paracrine growth factor considered to be important during embryogenesis; however, its expression by cells in the female reproductive tract has not been investigated. Therefore, an ovine FGF-10 cDNA was cloned from an ovine endometrial cDNA library to investigate expression and potential paracrine characteristics of FGF-10 in the ovine uterus. The ovine FGF-10 cDNA encodes a protein of 213 amino acids and possesses an unusually long 5' untranslated region (UTR). In situ hybridization demonstrated that ovine FGF-10 mRNA was expressed by endometrial stromal cells and by mesenchymal cells of the chorioallantoic placenta. The mRNA for FGF-7, a homologue of FGF-10, was localized in the tunica muscularis of blood vessels in endometrium and myometrium. In contrast, FGF receptor 2IIIb, the high-affinity receptor for both FGF-10 and FGF-7, was expressed exclusively in luminal epithelium, glandular epithelium, and placental trophectoderm. The in vivo spatial expression pattern suggests that FGF-10 is a novel endometrial stromal cell-derived mediator of uterine epithelial and conceptus trophectodermal functions. The nonoverlapping spatial patterns of expression for FGF-10 and FGF-7 in ovine uterus and conceptus suggest independent roles in uterine function and conceptus development.     

The chemokines, CX3CL1, CCL14, and CCL4, promote human trophoblast migration at the feto-maternal interface

Human embryo implantation is a complex process involving blastocyst attachment to the endometrial epithelium and subsequent trophoblast invasion of the decidua. Chemokines, critical regulators of leukocyte migration, are abundant in endometrial epithelial and decidual cells at this time. We hypothesized that endometrial chemokines stimulate trophoblast invasion. Chemokine receptors CX3CR1 and CCR1 were immunolocalized in human first-trimester implantation sites, specifically to endovascular extravillous trophoblasts, but not to the invading interstitial EVTs (iEVTs), with weak staining also on syncytium. CCR3 was localized to invading iEVTs and to microvilli on the syncytial surface. Expression of CX3CL1 (fractalkine), CCL7 (MCP-3), and their receptors (CX3CR1, CCR1, CCR2, CCR3, and CCR5) mRNA was examined in cellular components of the maternal-embryonic interface by RT-PCR. Both chemokines were abundant in entire endometrium and placenta, endometrial cells (primary cultures and HES, a human endometrial epithelial cell line) and trophoblast cell lines (JEG-3, ACIM-88, and ACIM-32). Chemokine receptor mRNA was expressed by placenta and trophoblast cell lines: CCR1 by all trophoblast cell types, whereas CCR2, CCR3, and CX3CR1 were more variable. CX3CR1, CCR1, CCR2, and CCR5 were also expressed by endometrial cells. Migration assays used the trophoblast cell line most closely resembling extravillous cytotrophoblast (AC1M-88). Trophoblast migration occurred in response to CX3CL1, CCL14, and CCL4, but not CCL7. Endometrial cell-conditioned media also stimulated trophoblast migration; this was attenuated by neutralizing antibodies to CX3CL1 and CCL4. Thus, chemokines are expressed by maternal and embryonic cells during implantation, whereas corresponding receptors are on trophoblast cells. Promotion of trophoblast migration by chemokines and endometrial cell conditioned medium indicates an important involvement of chemokines in maternal-fetal communication.     

Osteopontin is synthesized by uterine glands and a 45-kDa cleavage fragment is localized at the uterine-placental interface throughout ovine pregnancy

Osteopontin (OPN) is a phosphorylated and glycosylated, secreted protein that is present in various epithelial cells and biological fluids. On freezing and thawing or treatment with proteases, the native 70-kDa protein gives rise to 45- and 24-kDa fragments. Secreted OPN functions as an extracellular matrix (ECM) protein that binds cell surface receptors to mediate cell-cell adhesion, cell-ECM communication, and cell migration. In sheep and humans, OPN is proposed to be a secretory product of uterine glandular epithelium (GE) that binds to uterine luminal epithelium (LE) and conceptus trophectoderm to mediate conceptus attachment, which is essential to maintain pregnancy through the peri-implantation period. Cell-cell adhesion, communication, and migration likely are important at the interface between uterus and placenta throughout pregnancy, but to our knowledge, endometrial and/or placental expression of OPN beyond the peri-implantation period has not been documented in sheep. Therefore, the present study determined temporal and spatial alterations in OPN mRNA and protein expression in the ovine uterus between Days 25 and 120 of pregnancy. The OPN mRNA in total ovine endometrium increased 30-fold between Days 40 and 80 of gestation. In situ hybridization and immunofluorescence analyses revealed that the predominant source of OPN mRNA and protein throughout pregnancy was the uterine GE. Interestingly, the 45-kDa form of OPN was detected exclusively, continuously, and abundantly along the apical surface of LE, on conceptus trophectoderm, and along the uterine-placental interface of both interplacentomal and placentomal regions through Day 120 of pregnancy. The 45-kDa OPN is a proteolytic cleavage fragment of the native 70-kDa OPN, and it is the most abundant form in uterine flushes during early pregnancy. The 45-kDa OPN is more stimulatory to cell attachment and cell migration than the native 70-kDa protein. Collectively, the present results support the hypothesis that ovine OPN is a component of histotroph secreted by the uterine GE that accumulates at the uterine-placental interface to influence maternal-fetal interactions throughout gestation in sheep.     

Trophoblast-uterine interactions during equine chorionic girdle cell maturation, migration, and transformation.

The structure of the equine chorionic girdle between days 28 and 42 of gestation was examined. Of particular interest were differentiation of trophoblastic cells within the girdle, adhesion between girdle and endometrium, invasion and displacement of the uterine epithelium, and the nature of the endometrium when girdle cells migrate into it to form endometrial cup cells. The chorionic girdle, identified initially as a band of tall columnar cells, becomes a stratified columnar epithelium indented by clefts and pits. Adhesion to and penetration through the endometrial luminal epithelium are rapid and occur initially in very limited areas. Stromal invasion occurs as strands of contiguous trophoblast cells invade through the basal lamina. Only girdle cells that are adjacent to the basal lamina or have entered the endometrial stroma undergo hypertrophy and differentiate into cup cells. At the initiation of trophoblastic invasion, the luminal epithelium contains numerous, large, intraepithelial, granular lymphocytes; small lymphocytes then accumulate in the stroma, but by day 42 lymphocytes are largely confined to the periphery of the cup. Although adhesion of trophoblast to the endometrial surface is initiated by small groups of girdle cells on restricted areas of the endometrial folds, the area is then increased by new areas of adhesion and by expansion of the initial invasion. Areas of girdle cells that do not attach undergo necrosis, as do superficial portions of areas of invasion. Consequently the girdle cells that form cups may be a minority of the original population. It is suggested that the differentiation of girdle cells is closely programmed and that cells that do not reach the stroma become necrotic at the same time that endometrial cup cells are differentiating.     

The effects of cholera toxin, pertussis toxin, sodium fluoride and alpha-interferon on prostaglandin production by the guinea-pig endometrium

The outputs of prostaglandin (PG) F-2 alpha, 6-keto-PGF-1 alpha and PGE-2 from Day-7 and Day-15 guinea-pig endometrium were neither stimulated nor inhibited by cholera toxin and pertussis toxin. This indicates that PG synthesis by guinea-pig endometrium is not controlled by toxin-sensitive G-proteins. Short-term treatment of guinea-pig endometrium in culture with sodium fluoride stimulated PG output, suggesting that endometrial PG synthesis may be regulated by a fluoride-sensitive G-protein. Long-term treatment of guinea-pig endometrium in culture with sodium fluoride inhibited endometrial PG synthesis, and this was due to an inhibition of endometrial protein synthesis. Human alpha-interferon had no inhibitory effect on the outputs of PGF-2 alpha, 6-keto-PGF-1 alpha and PGE-2 from Day-15 guinea-pig endometrium in culture. It appears that the anti-luteolytic factor secreted by guinea-pig conceptus is not an alpha-interferon and is therefore probably different from ovine trophoblast protein-1.     

Analysis of osteopontin at the maternal-placental interface in pigs

Noninvasive, epitheliochorial placentation in the pig follows a prolonged preimplantation period characterized by migration, spacing and elongation of conceptuses, and secretion of estrogen for maternal recognition of pregnancy. Osteopontin (OPN) is an extracellular matrix protein that binds integrins to promote cell-cell attachment and communication. OPN appears to play a key role in conceptus implantation and maintenance of pregnancy in sheep; however, a role for OPN in the porcine uterus has not been established. Therefore, this study examined OPN expression and function in the porcine uterus and conceptus (embryo/fetus and associated extraembryonic membranes). Northern and slot blot hybridization detected an increase in endometrial OPN expression between Days 25 and 30, and levels remained elevated through Day 85 of pregnancy. In situ hybridization localized OPN mRNA to discrete regions of the uterine luminal epithelium (LE) on Day 15 of pregnancy and to the entire LE thereafter. Glandular epithelial (GE) expression of OPN mRNA was first detected on Day 35 of pregnancy and increased through Day 85. Both 70- and 45-kDa forms of OPN protein were detected in cyclic and pregnant endometrium by Western blotting. OPN protein was localized to the LE and GE by immunofluorescence; however, only the 70-kDa OPN was detected in uterine flushings. OPN protein was present along the entire uterine-placental interface after Day 30 of pregnancy. In addition, OPN mRNA and protein were localized to immune-like cells within the stratum compactum of the endometrium in both Day 9 cyclic and pregnant gilts. Incubation of OPN-coated microbeads with porcine trophectoderm and uterine luminal epithelial cells induced Arg-Gly-Asp (RGD)-dependent integrin activation and transmembrane accumulation of cytoskeletal molecules at the apical cell surface as assessed by immunofluorescence detection of talin or alpha-actinin as markers for focal adhesions. These results suggest that OPN, expressed by uterine epithelium and immune cells, may interact with receptors (i.e., integrins) on conceptus and uterus to promote conceptus development and signaling between these tissues as key contributors to attachment and placentation in the pig.     

Gastrin-releasing peptide (GRP) in the ovine uterus: regulation by interferon tau and progesterone

Gastrin-releasing peptide (GRP) is abundantly expressed by endometrial glands of the ovine uterus and processed into different bioactive peptides, including GRP1-27, GRP18-27, and a C-terminus, that affect cell proliferation and migration. However, little information is available concerning the hormonal regulation of endometrial GRP and expression of GRP receptors in the ovine endometrium and conceptus. These studies determined the effects of pregnancy, progesterone (P4), interferon tau (IFNT), placental lactogen (CSH1), and growth hormone (GH) on expression of GRP in the endometrium and GRP receptors (GRPR, NMBR, BRS3) in the endometrium, conceptus, and placenta. In pregnant ewes, GRP mRNA and protein were first detected predominantly in endometrial glands after Day 10 and were abundant from Days 18 through 120 of gestation. Treatment with IFNT and progesterone but not CSH1 or GH stimulated GRP expression in the endometrial glands. Western blot analyses identified proGRP in uterine luminal fluid and allantoic fluid from Day 80 unilateral pregnant ewes but not in uterine luminal fluid of either cyclic or early pregnant ewes. GRPR mRNA was very low in the Day 18 conceptus and undetectable in the endometrium and placenta; NMBR and BRS3 mRNAs were undetectable in ovine uteroplacental tissues. Collectively, the present studies validate GRP as a novel IFNT-stimulated gene in the glands of the ovine uterus, revealed that IFNT induction of GRP is dependent on P4, and found that exposure of the ovine uterus to P4 for 20 days induces GRP expression in endometrial glands.     

Porcine conceptus proteins: antiviral activity and effect on 2',5'-oligoadenylate synthetase.

Interferon-like proteins synthesized by conceptuses of domestic ruminants inhibit luteolysis during early pregnancy. Although pig conceptuses secrete trophoblast interferons during the period of CL maintenance, estrogen is involved with maintenance of the CL. The principal purposes of this work were to confirm production of trophoblast interferons by porcine conceptuses and to compare the effect of trophoblast interferons on endometrium of pigs and cattle. When measured using Madin-Darby bovine kidney (MDBK) cells challenged with vesicular stomatitis virus, antiviral activity in uterine flushings from cyclic gilts was not detectable throughout the estrous cycle; however, in pregnant gilts, antiviral activity increased from undetectable amounts to 4-11 x 10(3) U on Days 14, 16, and 18. Porcine embryos in culture produced 1,100 U/embryo/ml/24 h. Porcine conceptus secretory proteins induced 2',5'-oligo(A) synthetase in MDBK cells and in endometrial explants of cows but had no measurable effect on 2',5'-oligo(A) synthetase activity of endometrial explants of pigs. Similarly, endometrial 2',5'-oligo(A) synthetase of pregnant pigs was unaffected in vivo during the period of maximal synthesis of conceptus secretory proteins. Porcine conceptus secretory proteins produced no detectable increase in serum antiviral activity or 2',5'-oligo(A) synthetase activity of blood mononuclear leukocytes in utero-ovarian venous blood. These results suggest that conceptus interferons of pigs play different roles in the establishment of pregnancy compared to their roles in ruminants.     

Regulation of expression and role of leukemia inhibitory factor and interleukin-6 in the uterus of early pregnant pigs

Cytokines, which are generally involved in the process of inflammation, may also play a critical role in conceptus implantation. We examined: (1) the expression profiles of leukemia inhibitory factor (LIF) and interleukin (IL)-6 mRNA and their protein content in the endometrium of cyclic and pregnant gilts on Days 10 to 18 after estrus; (2) the effect of conceptus-exposed medium on LIF and IL-6 synthesis in the endometrium; (3) the profiles of IL6R and LIFR mRNA expression in pig conceptuses collected on Days 10 to 18 of pregnancy; and (4) the effect of LIF and IL-6 on the attachment and proliferation of porcine trophoblast cells. The expression of LIF mRNA in the endometrium increased between Days 10 and 12 in both cyclic and pregnant gilts, and tended to be higher in Day 12 pregnant animals compared with nonpregnant ones. The LIF protein content in the uterine lumen peaked on Day 12 of pregnancy, and was higher than on Day 12 of the estrous cycle. Endometrial IL-6 mRNA expression was upregulated on Day 12 in pregnant gilts compared with nonpregnant animals. Moreover, a higher content of IL-6 protein was observed in pregnant than in cyclic gilts. The addition of conceptus-exposed medium resulted in up-regulation of LIF and IL6 mRNA expression, and increased IL-6 content in endometrial slices. In conceptuses, increased mRNA expression was detected on Days 10 to 14 for IL6R and on Day 14 for LIFR, when compared with other days studied. LIF and IL-6 stimulated the attachment and proliferation of trophoblast cells in vitro. In summary, LIF and IL-6 are important components of embryo-uterine interactions during early pregnancy in the pig, and may contribute to successful conceptus implantation.     

Muc-1, integrin, and osteopontin expression during the implantation cascade in sheep

The extracellular matrix protein osteopontin (OPN) is a component of histotroph that increases in uterine flushings from pregnant ewes during the peri-implantation period and is localized on the apical surfaces of the uterine luminal epithelium (LE) and conceptus trophectoderm (Tr). The potential involvement of OPN in the implantation adhesion cascade in sheep was investigated by examining temporal, spatial, and potential functional relationships between OPN, Muc-1, and integrin subunits during the estrous cycle and early pregnancy. Immunoreactive Muc-1 was highly expressed at the apical surfaces of uterine luminal (LE) and glandular epithelium (GE) in both cycling and pregnant ewes but was decreased dramatically on LE by Day 9 and was nearly undetectable by Day 17 of pregnancy when intimate contact between LE and Tr begins. In contrast, integrin subunits alpha(v), alpha(4), alpha(5), beta(1), beta(3), and beta(5) were constitutively expressed on conceptus Tr and at the apical surface of uterine LE and GE in both cyclic and early pregnant ewes. The apical expression of these subunits could contribute to the apical assembly of several OPN receptors including the alpha(v)beta(3), alpha(v)beta(1), alpha(v)beta(5), alpha(4)beta(1), and alpha(5)beta(1) heterodimers on endometrial LE and GE, and conceptus Tr in sheep. Functional analysis of potential OPN interactions with conceptus and endometrial integrins was performed on LE and Tr cells in vitro using beads coated with OPN, poly-L-lysine, or recombinant OPN in which the Arg-Gly-Asp sequence was replaced with RGE or RAD. Transmembrane accumulation of talin or alpha-actinin at the apical surface of uterine LE and conceptus Tr cells in contact with OPN-coated beads revealed functional integrin activation and cytoskeletal reorganization in response to OPN binding. These results provide a physiological framework for the role of OPN, a potential mediator of implantation in sheep, as a bridge between integrin heterodimers expressed by Tr and uterine LE responsible for adhesion for initial conceptus attachment.     

IL-22 is expressed by the invasive trophoblast of the equine (Equus caballus) chorionic girdle

The invasive trophoblast cells of the equine placenta migrate into the endometrium to form endometrial cups, dense accumulations of trophoblast cells that produce equine chorionic gonadotropin between days 40 and 120 of normal pregnancy. The mechanisms by which the trophoblast cells invade the endometrium while evading maternal immune destruction are poorly defined. A gene expression microarray analysis performed on placental tissues obtained at day 34 of gestation revealed a >900-fold upregulation of mRNA encoding the cytokine IL-22 in chorionic girdle relative to noninvasive chorion. Quantitative RT-PCR assays were used to verify high expression of IL-22 in chorionic girdle. Additional quantitative RT-PCR analysis showed a striking increase in IL-22 mRNA expression in chorionic girdle from days 32 to 35 and an absence of IL-22 expression in other conceptus tissues. Bioinformatic analysis and cDNA sequencing confirmed the predicted length of horse IL-22, which carries a 3' extension absent in IL-22 genes of humans and mice, but present in the cow and pig. Our discovery of IL-22 in the chorionic girdle is a novel finding, as this cytokine has been previously reported in immune cells only. IL-22 has immunoregulatory functions, with primary action on epithelial cells. mRNA of IL-22R1 was detected in pregnant endometrium at levels similar to other equine epithelia. Based upon these findings, we hypothesize that IL-22 cytokine produced by the chorionic girdle binds IL-22R1 on endometrium, serving as a mechanism of fetal-maternal communication by modulating endometrial responses to trophoblast invasion.