Judith R. Head

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

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−/−γc−/− (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 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−/−γc−/− 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, 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.

Regulation of interleukin-8 gene expression in human endometrial cells in culture

In this study, we investigated the regulation of interleukin-8 (IL-8) gene expression in separated endometrial stromal and epithelial cells of human endometrium. This research was conducted as part of an analysis of the role of these cells in regulating the recruitment of leukocytes to the endometrium. Well-characterized model systems were used to study the regulation of endometrial IL-8 gene expression, namely, stromal cells in monolayer culture after first passage and glandular epithelium in primary culture. The levels of IL-8 mRNA and the accumulation of immunoreactive IL-8 in the medium of endometrial stromal cells is culture increased in a time- and concentration-dependent manner upon treatment with IL-1 alpha, tumor necrosis factor-alpha, or serum. The effects of IL-1 alpha plus serum on IL-8 mRNA levels were at least additive. Serum treatment caused a modest stimulation of IL-8 gene transcription (evaluated after 6 h of treatment) in endometrial stromal cells, but serum also acted in these stromal cells to prolong the half-life of IL-8 mRNA by more than 2.5-fold. The regulation of the levels of IL-8 mRNA in endometrial epithelial cells is distinctly different from that in stroma. First, the levels of IL-8 mRNA in non-treated epithelial cells in serum-free medium were much greater than those in stromal cells under similar conditions. Second, whereas the levels of IL-8 mRNA in endometrial epithelial cells also increased in response to serum and to IL-1 in the absence of serum, in the presence of serum, IL-1 treatment caused no appreciable change in the levels of IL-8 mRNA as was the case in endometrial stromal cells.

Uterine natural killer cells are targets for a trophoblast cell-specific cytokine, prolactin-like protein A

PRL-like protein A (PLP-A) is a member of the PRL family expressed in trophoblast cells coincident with establishment of the chorioallantoic placenta. The purpose of this investigation was to identify targets for PLP-A. Using an alkaline phosphatase-tagging strategy, we show that PLP-A specifically interacts with a population of natural killer (NK) lymphocytes within the mesometrial compartment of decidua from pregnant and pseudopregnant rats. These observations are supported by the codistribution of PLP-A targets with cells expressing the rat NK cell surface marker, gp42, the absence of PLP-A binding in conceptuses from NK cell-deficient tge26 mice, and the specific interaction of PLP-A with a rat NK cell line, RNK-16. We have further demonstrated that PLP-A effectively suppresses RNK-16 cell cytolytic activities. Our results provide evidence for a new paradigm of embryonic-maternal communication involving a PLP-A signaling pathway between trophoblast cells and uterine NK lymphocytes.

Murine trophoblast resists cell-mediated lysis: II. Resistance to natural cell-mediated cytotoxicity

The susceptibility of murine trophoblast cells to natural cell-mediated cytotoxicity has been assessed. Primary short-term cultures of murine trophoblast cells isolated from 14-day placentas were found to be resistant to endogenous and interferon-activated natural killer (NK) cells and natural cytotoxic cells. That the relevant target structures are expressed on the surface of trophoblast cells and accessible to the effectors was demonstrated by their ability to inhibit the lysis of NK-sensitive target cells (YAC-1) in a dose-dependent manner. The lytic resistance of trophoblast cells was unaffected by neuraminidase treatment, inhibition of protein synthesis, or extending the assay time to 12 hr. Moreover, trophoblast cells were resistant to antibody-dependent cell-mediated cytotoxicity when coated with an alloantibody capable of mediating their lysis in the presence of heterologous complement. Neither the preincubation of effector cells in concentrated trophoblast culture supernatants nor the direct exposure of effectors to monolayers of trophoblast cells inhibited their NK lytic activity, indicating that the secretion of a suppressive factor or the direct inactivation of the NK cells was not responsible for the observed resistance to lysis. These observations, together with previous results showing the resistance of trophoblast to cytotoxic T cell-mediated lysis, reveal that murine trophoblast cells possess a resistance mechanism against several forms of cell-mediated lysis. This feature of trophoblast cells at the maternal-fetal interface is likely to play an important role in protecting the fetoplacental allograft from immune rejection.

Decidua-associated suppressor activity and viability of individual implantation sites of allopregnant C3H mice.

Release of soluble suppressor activity from individual implant site decidua of DBA/2-mated C3H/HeJ mice was measured on days 12.5-13.5 of pregnancy. Suppressor activity varied among sites and followed a distribution curve that was displaced towards low suppression when resorption sites were compared to healthy embryonic implants. Pre-immunization against the DBA/2 strain paternal antigens failed to increase resorption (by loss of low suppression implants) but led instead to a reduced resorption rate. This was associated with an increase in soluble suppressor activity obtained from decidua. Some reduction in resorption occurred independent of an increase in the level of suppression suggesting additional contributing factors to the immunization effect.

Can Trophoblast Be Killed by Cytotoxic Cells?: In Vitro Evidence and in Vivo Possibilities

It ha.s been decades since the important concept was formahzed that the placenta may represent an immunologically inert buffer, protecting both the fetus and itselffrom destruction by maternal effectors. Initially, the need for such a mechanism was rationalized by the an8:logy of the fetal-placental unit to an allograft gen~~1cally ~apable ~f expressing paternal histocompatibility antigens, WIth the potential for destruction by maternal alloantibodies or allospecific cytotoxic T lymphocytes (CTL).2 The focus of attention was quite naturally the trophoblast cells, the frontier fetal-derived cells at the maternal-fetal interface. Two key questions central to this issue were, 1) are the relevant antigens expressed on trophoblast cells? and 2) if so, are trophoblast cells susceptible to lysis by allospecific effectors? With the discoveries of several other cytotoxic cells such as those mediating various forms of natural, non~ MHC-restricted cytotoxicity, the questions have been expanded to include these cytotoxic effectors as well.

Murine trophoblast can be killed by allospecific cytotoxic T lymphocytes generated in GIBCO Opti-MEM medium

Previous work in this laboratory demonstrated that a population of cultured midterm murine trophoblast cells are not susceptible to lysis by allospecific cytotoxic T lymphocytes (CTL) generated by standard in vitro protocols. We now report that this trophoblast population is killed, in an MHC-dependent manner, by allospecific CTL generated in GIBCO Opti-MEM, a modified tissue culture medium designed to maintain cell growth and proliferation in the presence of low concentrations of fetal bovine serum (FBS).

Murine trophoblast cells are not killed by tumor necrosis factor-α

Purified midgestation murine trophoblast cannot be killed by a variety of cell-mediated effector mechanisms, with the exception of highly lytic effectors such as lymphokine-activated killer cells. We now report that this trophoblast population is also resistant to tumor necrosis factor (TNF)-alpha.

GM-CSF and CSF-1 stimulate DNA synthesis but not cell proliferation in short-term cultures of mid-gestation murine trophoblast

Short-term cultures of purified murine trophoblast were used to investigate the potential trophic effects of a number of cytokines. Both granulocyte-macrophage colony stimulating factor (GM-CSF) and colony stimulating factor-1 (CSF-1) increased [3H]thymidine (TdR) uptake (3-8 times control values) by trophoblast harvested from placentae on day 12 or 14 of pregnancy. In contrast, interleukin-3 (IL-3) had only a mild stimulatory effect ([3H]TdR uptake 1.5 times control), and IL-2 did not alter the level of DNA synthesis in these cells. Immunocytochemical analysis confirmed that the cells engaged in DNA synthesis were cytokeratin-positive trophoblast cells and revealed that these cells predominantly bore markers (alkaline phosphatase, transferrin receptors) characteristic of trophoblast cells from the placental labyrinth. The increased DNA synthesis observed after exposure to GM-CSF or CSF-1 was not associated with a change in the proportion of nuclei involved in synthesis, nor did it result in significantly increased trophoblast cell numbers in the cultures. These findings suggest that DNA-synthesizing trophoblast cells were not proliferating, but were more likely engaged in endoreduplicative cycles leading to the formation of terminally differentiated trophoblast giant cells. These results caution against the presumption of proliferation when measuring [3H]thymidine incorporation by placental or trophoblast cells in standard in vitro cultures. In addition, taken together with the reports of high levels of CSF-1 in the pregnant uterus and the expression of the CSF-1 receptor on placental trophoblast cells, they suggest that the hemopoietic cytokines may play a role in the differentiation and/or function of trophoblast cells in the developing murine placenta.

Culture characteristics of human endometrial glandular epithelium throughout the menstrual cycle: Modulation of deoxyribonucleic acid synthesis by 17β-estradiol and medroxyprogesterone acetate

OBJECTIVEnThe purpose of this study was to evaluate the culture characteristics and cell proliferation of human endometrial glandular epithelial cells in primary culture on extracellular matrix and to evaluate sex steroid modulation of this process.nnnSTUDY DESIGNnWe examined the culture characteristics in 53 endometrial gland preparations obtained throughout the menstrual cycle and determined the incorporation of tritiated thymidine in endometrial epithelial cells as a measure of deoxyribonucleic acid synthesis (cell proliferation) in the presence and absence of 17 beta-estradiol and medroxyprogesterone acetate.nnnRESULTSnGood culture maintenance of endometrial epithelial monolayers on extracellular matrix was observed from glands derived from proliferate phase endometrium (21 of 23), whereas poor adherence and culture maintenance was observed in all 30 of specimens from the secretory phase. With 17 beta-estradiol treatment for 22 hours, tritiated thymidine incorporation was not different from control, but medroxyprogesterone acetate treatment for 22 hours was associated with diminished tritiated thymidine incorporation by 36% (p < 0.004). When 17 beta-estradiol (10(-8) mol/L) was included in the incubation medium from the time of initial culture, tritiated thymidine incorporation on day 4 was 40% of control (p < 0.006), and tritiated thymidine was not suppressed further by 22 hours of treatment with medroxyprogesterone acetate (10(-7) mol/L). In spite of cellular spread of endometrial epithelial cells to subconfluence, deoxyribonucleic acid content per well did not increase over time in culture.nnnCONCLUSIONSnWe conclude that exposure of endometrial glands to progesterone in vivo inhibits adherence and the establishment of cell monolayers cultured on a thin layer of extracellular matrix. Because 17 beta-estradiol treatment in culture for 22 hours does not increase tritiated thymidine incorporation, it is possible that 17 beta-estradiol exerts its proliferative effect on the endometrial epithelium by an indirect action mediated by stromal cells. The effects of 17 beta-estradiol and medroxyprogesterone acetate on deoxyribonucleic acid synthesis in endometrial epithelial cells may represent an action on a stem cell population of dividing cells or a terminally differentiated cell population that undergoes programmed cell death.