Ephrin-B2-expressing natural killer cells induce angiogenesis.

Background: Therapeutic angiogenesis aims to induce new blood vessel growth in ischemic tissues; however, previous clinical trials have had limited success. Studies of uterine angiogenesis revealed a specialized subset of natural killer (NK) cells, called uterine NK (uNK) cells, which have unique proangiogenic abilities. Methods: We show that uNK cells in mice express ephrin-B2, a regulator of angiogenesis, to induce tubule formation in an ex vivo coculture tubule formation assay. We next induced the expression of ephrin-B2 by splenic NK (sNK) cells harvested from male mice. Results: We showed that induced NK (iNK) cells can also instruct endothelial cells to form tubules using ephrin-B2. Conclusions: We concluded that Ephrin-B2 is a marker of proangiogenic uNK cells and that a proangiogenic phenotype characterized by ephrin-B2 can be induced in sNK cells to induce therapeutic angiogenesis.

Dysregulated uterine natural killer cells and vascular remodeling in women with recurrent pregnancy losses.

PROBLEM: Angiogenesis and vascular remodeling in secretory endometrium represent one of the crucial steps in pregnancy establishment, for which uterine NK (uNK) cells have an important role. Impairment of these steps may proceed to implantation and instigate initial pathology of recurrent pregnancy losses (RPL). In this study, we aim to investigate vascular development and density of uNK cells in secretory endometrium of women with RPL. METHODS OF STUDY: Mid-secretory phase endometrial tissues from women with RPL (n = 15) and fertile controls (n = 7) were investigated. CD56+ and CD16+ uNK cells, CD31+ vascular endothelial cells and smooth muscle myosin (SMM)+ . Vascular smooth muscle cells (VSMC) expressing SMM were investigated using immunohistochemistry and western blot. High-throughput quantitative real-time polymerase chain reaction (qRT-PCR) was used as well. RESULTS: CD56+ uNK number was significantly higher in women with RPL compared to controls (P < 0.0001). uNK cell density by immunohistochemistry was positively correlated with CD56 mRNA expression by qRT-PCR (r2  = 0.43, P = 0.0137). The number of blood vessels represented by the expression of either CD31 or SMM was higher in women with RPL as compared to controls (P < 0.05 and P < 0.0001, respectively), and correlated with the number of uNK cell (r2  = 0.18, P < 0.04, and r2  = 0.65, P < 0.0001, respectively). The wall thickness of spiral arteries was significantly higher in women with RPL as compared with that of controls (P = 0.0027). CONCLUSION: Increased uNK cells in mid-secretory endometrium are associated with increased vascularization and defective vascular transformation of spiral arteries in women with RPL.

Prevalence of HHV-6 in endometrium from women with recurrent implantation failure.

PROBLEM: To study the prevalence of HHV-6 in endometrial biopsies among women experiencing recurrent implantation failure (RIF) after IVF/ET compared with controls. METHOD OF STUDY: Thirty women experiencing RIF after IVF/ET and 10 fertile women participated in the study. All women had endometrial biopsies taken in the luteal phase of their menstrual cycle for an endometrial immune profile (EIP) and HHV-6 mRNA as well as lymphocyte and granulocyte populations. The prevalence of HHV-6 in endometrial biopsies was determined, and biopsies for positive and negative expression of HHV-6 were compared with the results of their EIP and lymphocyte and granulocyte populations. RESULTS: Thirty-seven percentage of women with a history of RIF and 0% of controls demonstrated the presence of HHV-6 in their endometrial biopsies. No associations were found when the results of the endometrial immune profile were compared with the presence or absence of HHV-6. Significant increase in neutrophil-specific CD16b mRNA was found in HHV-6-positive samples, and the levels of B cells-related CD19 mRNA were lower in biopsies from women with RIF in comparison with normal controls. CONCLUSION: HHV-6 infection is an important factor in RIF.

Predicting NK cell subsets using gene expression levels in peripheral blood and endometrial biopsy specimens.

PROBLEM: In molecular analysis of tissue biopsy specimens, one crucial aspect is characterization of immune cell populations. This is especially important for evaluation of uterine receptivity by assessing levels of lymphocyte populations including CD56bright CD16- uterine NK cells and CD56dim CD16+ conventional NK cells. Our objective was to investigate whether measuring total RNA transcripts from a tissue specimen would accurately reflect immune cell levels and be a new technique to assess immune cell subsets. METHOD OF STUDY: Peripheral blood mononuclear cells (PBMCs) and endometrial tissues were used. Flow cytometry was utilized for the analysis of lymphocyte subsets in PBMCs, and RT-qPCR was applied to quantify RNA transcripts indicative of lymphocyte and granulocyte populations. RESULTS: In PBMC specimens, there were significant correlations between gene expression levels and cell subsets. NK cells correlated with CD16A, NKp46, and CD56 transcripts, B cells correlated with EBF1, and CD8+ T cells correlated with CD8ß. Finally, endometrial tissues displayed high CD56 expression and very low CD3ε, CD16A, and NKp30, reflecting the characteristic endometrial NK cell subsets. CONCLUSION: Strong correlations between RT-qPCR data and levels of lymphocyte subsets indicate that gene expression analysis will be a useful technique for characterizing levels of CD56+ cells in endometrial tissues.

Recurrent Pregnancy Loss in Women with Killer Cell Immunoglobulin-Like Receptor KIR2DS1 is Associated with an Increased HLA-C2 Allelic Frequency.

PROBLEM: During human pregnancy, the uterine lining is highly populated with killer-immunoglobulin-like receptor (KIR)-expressing NK cells that recognize HLA-C molecules on trophoblast cells. The goal of this study was to analyze the KIR gene contents and frequencies in a N. American cohort of women with RPL of unknown etiology to evaluate whether there is a genetic susceptibility to RPL based on a woman's KIR repertoire and her HLA-C group, as well as the HLA-C group of the partner. METHOD OF STUDY: The frequencies of KIR and HLA-C1 and HLA-C2 genes were evaluated in 139 Caucasian women with RPL; HLA-C1, and HLA-C2 group genes were analyzed in their partners (n = 42). The gene frequencies were compared with data reported from corresponding populations. RESULTS: Overall, the frequencies of HLA-C groups and KIR genes and genotypes in RPL cohort resembled the frequencies for US Caucasians. The HLA-C1 and HLA-C2 group distribution was significantly different between women with or without KIR2DS1. Women positive for KIR2DS1 (45.3% of the study cohort) had an increased frequency of its ligand, HLA-C2 (0.5159 versus 0.3684 in KIR2DS1 negative women, P = 0.014). CONCLUSION: Our results indicate that among KIR2DS1 pos women, the co-expression of HLA-C2 is associated with RPL.

Immunophenotype and cytokine profiles of rhesus monkey CD56bright and CD56dim decidual natural killer cells.

The primate endometrium is characterized in pregnancy by a tissue-specific population of CD56(bright) natural killer (NK) cells. These cells are observed in human, rhesus, and other nonhuman primate decidua. However, other subsets of NK cells are present in the decidua and may play distinct roles in pregnancy. The purpose of this study was to define the surface marker phenotype of rhesus monkey decidual NK (dNK) cell subsets, and to address functional differences by profiling cytokine and chemokine secretion in contrast with decidual T cells and macrophages. Rhesus monkey decidual leukocytes were obtained from early pregnancy tissues, and were characterized by flow cytometry and multiplex assay of secreted factors. We concluded that the major NK cell population in rhesus early pregnancy decidua are CD56(bright) CD16(+)NKp30(-) decidual NK cells, with minor CD56(dim) and CD56(neg) dNK cells. Intracellular cytokine staining demonstrated that CD56(dim) and not CD56(bright) dNK cells are the primary interferon-gamma (IFNG) producers. In addition, the profile of other cytokines, chemokines, and growth factors secreted by these two dNK cell populations was generally similar, but distinct from that of peripheral blood NK cells. Finally, analysis of multiple pregnancies from eight dams revealed that the decidual immune cell profile is characteristic of an individual animal and is consistently maintained across successive pregnancies, suggesting that the uterine immune environment in pregnancy is carefully regulated in the rhesus monkey decidua.

Modulation of cytokine and chemokine secretions in rhesus monkey trophoblast co-culture with decidual but not peripheral blood monocyte-derived macrophages.

PROBLEM: Decidual macrophages are thought to promote pregnancy success, in part through interactions with invading trophoblast cells in hemochorial placentation. However, the factors that constitute this regulatory cross talk are not well understood. METHOD OF STUDY: Rhesus monkey decidual and peripheral blood-derived macrophages were co-cultured with primary Rhesus trophoblasts. Macrophage functions including cell-surface marker expression, antigen uptake and processing, in vitro migration, and cytokine and chemokine secretions were evaluated. RESULTS: While most macrophage functions were unchanged by trophoblast co-culture, changes in the secretion of selected cytokines and the migration of trophoblasts were noted when decidual (but generally, not peripheral blood monocyte-derived) macrophages were cultured with trophoblasts. In addition, basal secretion differed significantly between peripheral blood-derived and decidual macrophages for a broad spectrum of cytokines. When trophoblasts were pre-treated with an anti-Mamu-AG antibody, 25D3, there was no change in cytokine or chemokine secretion. CONCLUSION: Macrophage cytokine expression can be modulated by trophoblast co-culture, but it remains unclear how Mamu-AG is involved.

Placental-derived mesenchyme influences chorionic gonadotropin and progesterone secretion of human embryonic stem cell-derived trophoblasts.

Studies of early placental development in humans are difficult because of limitations on experimental material availability from the perimplantation period. We used a coculture system to determine the effects of various effector cell types on trophoblast differentiation. Enhanced green fluorescent protein (EGFP)-expressing H1 human embryonic stem cells were used in co-suspension with human term placental fibroblasts (TPFs) and dermal fibroblasts (CI2F) to form combination embryoid bodies (EBs), with the goal of recapitulating placental morphogenesis through incorporation of placental mesenchymal cells. Overall, the results demonstrated that when using mesenchymal cells for EB preparation from term placentas (TPF), combination EB-derived trophoblasts secrete higher levels of human chorionic gonadotropin (hCG) and progesterone compared to EBs made without effector cells, whereas there was no effect of dermal fibroblasts. This is due to the secretory activity of the EB-derived trophoblasts and not due to the number of differentiated trophoblasts per EB, demonstrating that nontrophoblast cells of the placenta can influence trophoblast endocrine activity.

On the role of placental Major Histocompatibility Complex and decidual leukocytes in implantation and pregnancy success using non-human primate models.

While there is broad agreement that interactions of the human maternal immune system with the tissues and cells of the implanting embryo are likely to be critical contributors to pregnancy success, there remains a dearth of information which directly confirms this expectation. Although animal models of reproductive function often provide opportunities for confirming such hypotheses, progress in this area has been sporadic due to limitations of traditional laboratory or agricultural animal models, such as rodents, sheep, pigs and cattle. Many of these limitations derive from divergent modes of implantation and placentation across mammalian species. Over the past decade there has been progress in the development of the nonhuman primate as a model in which to address questions of pregnancy success in the area of immunology. The purpose of this review is to compare available model species, summarize current knowledge and recent progress with an emphasis on experimental in vivo manipulations, and suggest areas available for additional study and growth.

Generation of macrophages from peripheral blood monocytes in the rhesus monkey.

Macrophages are found in tissues throughout the body and are important immune cells, however, these tissue macrophages are difficult to collect and study. Therefore, the ability to differentiate macrophages from peripheral blood precursors is an important research tool. Macrophage differentiation has been well studied in humans, but differentiation in the non-human primate is poorly characterized. Using human models is not always feasible for invasive experimental studies and, therefore, developing reliable protocols for the non-human primate model is important. We describe a method to differentiate macrophages in vitro in the rhesus monkey by culturing adherent peripheral blood mononuclear cells for five days in RPMI-1640 supplemented with 1% human serum, M-CSF, and IL-1beta. The resulting cells had a distinct macrophage phenotype, the ability to secrete cytokines in response to LPS, and antigen uptake and processing capabilities.

Characterization of cynomolgus and vervet monkey placental MHC class I expression: diversity of the nonhuman primate AG locus.

Nonhuman primates are important animal models for the study of the maternal immune response to implantation within the decidua. The objective of this study was to define the placental expression of major histocompatibility complex (MHC) class I molecules in the cynomolgus (Macaca fascicularis) and vervet (African green) (Chlorocebus aethiops) monkeys. Early pregnancy (d36-42) cynomolgus and vervet placentas were obtained by fetectomy and prepared for histological evaluation. A pan-MHC class I monoclonal antibody demonstrated MHC class I expression in both vervet and cynomolgus placental trophoblasts, with particularly high expression in the villous syncytium, as previously shown in the rhesus and baboon. Placental cytotrophoblasts were isolated by enzymatic dispersion and gradient centrifugation and cultured, and multicolor flow cytometry was used to phenotype cell populations. Culture of isolated villous cytotrophoblasts demonstrated that MHC class I expression was linked to syncytiotrophoblast differentiation. A monoclonal antibody against Mamu-AG, the nonclassical MHC class I homolog of HLA-G in the rhesus monkey, demonstrated intense immunostaining and cell surface expression in cynomolgus placental trophoblasts; however, staining with vervet placenta and cells was low and inconsistent. Reverse transcriptase polymerase chain reaction was used to clone MHC class I molecules expressed in cynomolgus and vervet placentas. While Mafa-AG messenger RNA (mRNA) was readily detectable in cynomolgus placental RNA and was >99% identical at the amino acid level with Mamu-AG, 7/8 Chae-AG complementary DNAs had an unusual 16 amino acid repeat in the alpha1 domain, and all clones had an unexpected absence of the early stop codon at the 3'-end of the mRNA diagnostic for rhesus, cynomolgus, and baboon AG mRNAs, as well as HLA-G. We conclude that while the vervet monkey has retained the placental expression of a primate-specific nonclassical MHC class I locus, diversity is also revealed in this locus expressed at the maternal-fetal interface, thought to participate in placental regulation of the maternal immune response to embryo implantation and pregnancy.

Suppression of Mamu-AG by RNA interference.

PROBLEM: The role of placental major histocompatibility complex (MHC) class I molecules in pregnancy is not well understood. Mamu-AG, the rhesus monkey homology of human leukocyte antigen (HLA)-G expressed in the human placenta, was targeted for degradation by RNA interference (RNAi), a powerful tool to aid in determining gene function, to determine the effect that this knockdown has on NK cell function. METHOD OF STUDY: A series of potential target short hairpin RNA (shRNA) sequences to suppress Mamu-AG expression was screened, which identified an optimal sequence to use in transfection experiments. Knockdown in two different Mamu-AG-expressing cell lines was measured by flow cytometry. Cytotoxicity assays were performed to correlate Mamu-AG expression with NK cell cytotoxicity. RESULTS: Decreased expression of Mamu-AG by short interfering RNA (siRNA) (70-80%) in cell types tested was associated with increased lysis of Mamu-AG target cells. CONCLUSION: Target sequences have been identified that knocked down Mamu-AG expression by RNAi and increased lysis by NK cells. This supports the concept that NK cell receptors recognize this placental non-classical MHC class I molecule.

Characterization of decidual leukocyte populations in cynomolgus and vervet monkeys.

The objective of this study was the phenotypic and functional evaluation of decidual immune cells in the cynomolgus and vervet monkeys. Early pregnancy (days 36-42) deciduas were obtained by fetectomy for histological evaluation and decidual mononuclear leukocyte (MNL) isolation. While peripheral NK (pNK) cells in these species do not express CD56, CD56(+) NK cells were abundant in decidual samples. The majority of decidual NK (dNK) cells (>80%) had high light-scatter characteristics and were CD56(bright)CD16(+) cells with no or very low levels of natural cytotoxicity receptors (NKp46, NKp30) and NKG2A, while a minor population were small CD56(dim)CD16(-) lymphocytes also expressing less NKp46, NKp30 and NKG2A than pNK cells. All dNK cells were found to be perforin(+); however, their cytotoxic potential was low and cynomolgus dNK cells showed strongly reduced cytotoxicity against target cells compared with pNK cells. Macrophages and T cells together comprised approximately 25-30% of decidual MNL. Decidual T cells contained a higher proportion of the minor T cell subtypes (gammadeltaT cells, CD56(+) T cells) compared with peripheral blood. A subset of DC-SIGN(+) macrophages, with a distribution adjacent to areas of placental attachment in contrast to the widespread setting of general CD68(+) cells, was identified in both species. Together, these results demonstrate that the maternal-fetal interface in both cynomolgus and vervet monkeys is very rich in immune cells that have similar phenotypes to those seen in humans, indicating that both species are excellent models to study the contributions of distinct immune cell populations to pregnancy support.

Immune and trophoblast cells at the rhesus monkey maternal-fetal interface.

To promote the use of the nonhuman primate model for the study of the cellular and molecular biology of maternal-fetal interactions and placental development during early pregnancy, we have developed protocols for the isolation and characterization of placental trophoblasts and decidual immune cells from the rhesus monkey. In this chapter, we provide protocols for trophoblast and decidual immune cell isolation, phenotyping of isolated cells by flow cytometry, and analysis of placental and decidual tissues by immunohistochemistry. Information on antibodies for these analyses are also provided, which is an important consideration when attempting to use anti-human antibodies for the study of nonhuman primates.

Stable plasmid-based siRNA silencing of gene expression in human embryonic stem cells.

RNA interference (RNAi) using short inhibitory RNAs (siRNAs) has been widely explored for the suppression of cellular mRNA levels to investigate the function of specific genes, including gene function in differentiation and development. The establishment of human embryonic stem cell (hESC) models for differentiation of selected lineages is an area of intense interest and activity. On the basis of our previous work with stable overexpression of enhanced green fluorescent protein (EGFP) in hESC, we used plasmid vector-based siRNA expression to silence EGFP expression in stably-transfected hESC. After hygromycin selection, we derived several cell lines in which EGFP expression was significantly reduced. At the genomic DNA level, there was no difference between the two cell lines and the parental H1EGFP cell line when analyzed with quantitative PCR; however, there were significant differences among the three cell lines at the RNA and protein levels as analyzed with real-time RT-PCR and Western blotting. From these data, we conclude that the decrease in EGFP expression was caused by RNAi, not by genomic DNA loss. Down-regulation of EGFP expression was sustained through multiple passages of both siEGFP cell lines. This simple silencing system will allow novel investigations of target gene function in hESC self-renewal or differentiation, as well as differentiated function in other cell types.

Study of interaction between the polyoxidonium immunomodulator and the human immune system cells.

Polyoxidonium (PO) is a high-molecular weight physiologically active compound with pronounced immunomodulating activity, an N-oxidized polyethylene-piperazine derivative. The aim of our work was to study cellular and molecular mechanisms of the action of PO on the human peripheral blood leukocytes. By means of flow cytometry it was established that the binding of fluorescein-isothiocyanate-labeled PO (FITC-labeled PO) occurs more rapidly with monocytes and neutrophils than with lymphocytes (7- to 8-fold weaker as compared with monocytes). Using colloidal gold-labeled PO and electron microscopy it was shown with that the preparation penetrates into leukocytes by endocytosis. PO is localized in endoplasmic vesicles of cellular cytosol. Analysis of one of the crucial signal transducer, the intracellular Ca(2+), performed with the Fluo-3 fluorescent dye, showed that PO does not induce Ca(2+) mobilization from the intracellular calcium stores and influx of extracellular Ca(2+). The study of the intracellular hydrogen peroxide (H(2)O(2)) production with the 2',7'-dichlorfluorescein indicator demonstrated that PO significantly increases the level of intracellular H(2)O(2) in monocytes and neutrophils, however, this increase is much less as compared with phorbol myristate acetate stimulation. The analysis of immunomodulating effect produced by PO proved its stimulating activity on some cytokines production in vitro, e.g. interleukin 1beta (IL-1beta), tumor necrosis factor (TNF)-alpha and IL-6. A dose-dependent increase in the intracellular killing by blood phagocytes was established under the action of PO.

Maintenance of pluripotency in human embryonic stem cells stably over-expressing enhanced green fluorescent protein.

The availability of human embryonic stem (HES) cells with a readily evaluated genetic marker such as green fluorescent protein (GFP) could facilitate a number of experimental opportunities. We constructed a novel plasmid with two elongation factor-1alpha (EF-1alpha) promoters (YPL2) to obtain a vector with mammalian promoters for simultaneous transgene expression in HES cells. An enhanced green fluorescent protein (EGFP) cDNA was inserted under the control of the first EF-1alpha promoter to construct plasmid YPL2-EGFP. The second EF1-alpha promoter was upstream of the neomycin resistance gene. H1 HES cells were transfected with YPL2-EGFP using Fugene 6. Following 100 microg/ml neomycin selection, individual colonies demonstrating stable EGFP expression were observed. After 4 months of passage under neomycin selection, the cells continued to maintain typical HES cell morphology. Undifferentiated cells showed no change in EGFP expression as determined by FACS analysis. Immunostaining demonstrated maintenance of Oct-3/4 expression in undifferentiated H1EGFP cells that was indistinguishable from wild-type HES cells. Addition of 10 ng/ml bone morphogenic protein-4 (BMP-4) to the cells provoked morphological and functional differentiation to trophoblasts, but no loss of EGFP expression. Following injection of EGFP-HES cells into immunodeficient mice, there was robust formation of teratomas that demonstrated a broad range of morphological pluripotency with widespread EGFP expression. EGFP expression was also maintained in differentiating embryoid bodies formed from EGFP-HES cells. This report demonstrates that ES cells carrying EGFP will be useful in diverse areas of embryonic stem cell research.

Effect of polyoxidonium on the phagocytic activity of human peripheral blood leukocytes.

The effect of polyoxidonium on the functional activity of human peripheral blood phagocytes was studied in vitro. Polyoxidonium is an N-oxidized polyethylene-piperazine derivative, a water-soluble high-molecular synthetic immunomodulator. It was established that a one-hour incubation of leukocytes with polyoxidonium increases the ability of leukocytes to kill the ingested Staphylococcus aureus in a dose-dependent manner. This increase was observed in leukocytes obtained from healthy individuals and from patients with chronic granulomatous disease. The study of phagocyte spontaneous and stimulated chemiluminescence showed a significant decrease in the quantity of chemiluminescent impulses in the extracellular space in the presence of polyoxidonium both in luminol- and lucigenin-dependent chemiluminescence assays. Polyoxidonium proved to have an antioxidant activity at all doses tested (100, 250, and 500 &mgr;g/ml). Evaluation of the intracellular hydrogen peroxide (H(2)O(2)) level with a fluorescent indicator dichlorofluorescein showed that incubation with polyoxidonium leads to a higher luminescence intensity of dichlorofluorescein, thus indicating an increase in the intracellular H(2)O(2) level. This increase was not as substantial as in the case of stimulation with phorbol myristate acetate. When polyoxidonium was used at a dose of 500 &mgr;g/ml, the difference with the control was significant for neutrophils and monocytes. Polyoxidonium can be used as adjuvant in combined treatment of acute and chronic infections of any etiology, and in the treatment of chronic granulomatous disease and secondary immunodeficiences along with etiotropic drugs.