Larry J. Guilbert

Bidirectional cytokine interactions in the maternal-fetal relationship: is successful pregnancy a TH2 phenomenon?

Pregnant females are susceptible to intracellular pathogens and are biased towards humoral rather than cell-mediated immunity. Since TH1 cytokines compromise pregnancy and TH2 cytokines are produced at the maternal-fetal interface, we hypothesize that these TH2 cytokines inhibit TH1 responses, improving fetal survival but impairing responses against some pathogens.

Cytotoxicity of tumour necrosis factor-alpha and gamma-interferon against primary human placental trophoblasts

Tumour necrosis factor-alpha (TNF-alpha) and gamma interferon (IFN-gamma) are expressed within human placental villi during normal pregnancy, yet their functions remain unknown. Since villous cytotrophoblasts are within the paracrine reach of this expression, the effects of TNF-alpha and IFN-gamma on a purified population of term placental cytotrophoblasts were examined. After 4 days of culture TNF-alpha alone induced a loss of trophoblast viability as measured by both metabolic capacity (MTT reduction) and DNA content. The combination of TNF-alpha and IFN-gamma enhanced the damaging effect. Neutralizing antibodies against TNF receptor p55, but not p75, partially reversed the TNF-alpha-induced cytotoxicity. After 24 h of culture, TNF-alpha and IFN-gamma increased the fraction trophoblasts containing nicked DNA, and after 60 h, increased the detachment of cells characterized by a distorted morphology, lower DNA content, and fragmented DNA. These results suggest that a physiological role of TNF-alpha and IFN-gamma expression in the placental villi may be to regulate the apoptotic death of villous cytotrophoblasts. The studies also predict potential harmful effects on placental development and function following aberrant inflammatory cytokine expression triggered by intravillous infections.

Inflammatory processes in preterm and term parturition

A role for the pro-inflammatory cytokines interleukin (IL)-1beta, IL-6, IL-8 and tumor necrosis factor alpha (TNF-alpha) is evident in term and preterm delivery, and this is independent of the presence of infection. All uterine tissues progress through a staged transformation near the end of pregnancy that leads from relative uterine quiescence and maintenance of pregnancy to the activation of the uterus that prepares it for the work of labour and production of stimulatory molecules that trigger the onset of labour and delivery. The uterus is activated by pro-inflammatory cytokines through stimulation of the expression and production of uterine activation proteins (UAPs). One of these actions is the stimulation of prostaglandin (PG) synthesis. Particularly important for labour is PGF(2alpha) and its receptor, PTGFR. In addition, pro-inflammatory cytokines are able to increase the synthesis of matrix metalloproteinases (MMPs), vascular endothelial growth factor (VEGF) and the progesterone receptor C isoform, which leads to decreased tissue progesterone responsiveness. Some of these effects are replicated by PGF(2alpha), suggesting that it may act via its receptor to amplify the direct actions of cytokines. In turn, VEGF may enhance leukocyte recruitment to the uterus, and MMP-9 may promote activation of inactive pro-form cytokines. Pro-inflammatory cytokines also decrease the activity of 11beta-hydroxysteroid dehydrogenase, which likely increases intrauterine cortisol concentrations. In turn, cortisol may drive PG synthesis. Together these feed-forward mechanisms activate the uterus, trigger the production of uterine contractile stimulants and lead to labour and delivery.

Biology of the hemopoietic microenvironment.

Abstract: In adult mammals, hemopoiesis takes place primarily in the bone marrow. The steady‐state production of blood cells depends to a large extent on the interaction between hemopoietic stem/progenitor cells (HPC) and the different components of the microenvironment present in the medullary cavity. During the last three decades, in vivo and in vitro studies have allowed significant advances in understanding of the biology of such a hemopoietic microenvironment. Although not evident in histological sections, it is well known that the hemopoietic microenvironment is a highly organized structure that regulates the location and physiology of HPC. The hemopoietic microenvironment is composed of stromal cells (fibroblasts, macrophages, endothelial cells, adipocytes), accessory cells (T lymphocytes, monocytes), and their products (extracellular matrix and cytokines). Microenvironmental cells can regulate hemopoiesis by interacting directly (cell‐to‐cell contact) with HPC and/or by secreting regulatory molecules that influence, in a positive or negative manner, HPC growth. Recent in vitro studies suggest that functional abnormalities of the hemopoietic microenvironment may be implicated in the manifestation of certain hematological disorders such as aplastic anemia, and acute and chronic myelogenous leukemia. Thus, the characterization of the structure and function of the human hemopoietic microenvironment may have relevance in understanding and treating different hematological disorders.

Epidermal growth factor inhibits cytokine-induced apoptosis of primary human trophoblasts

In the placenta, as in other organs, the development and maintenance of the differentiated phenotype depend on a balance between cell proliferation, maturation, and death. We are interested in the mechanisms that regulate the survival and differentiation of placental trophoblasts and have recently demonstrated that the inflammatory cytokines tumor necrosis factor alpha (TNFα) and gamma interferon (IFNγ) act in concert to induce apoptotic cell death in normal cytotrophoblasts in culture. In this report we show that exposure to epidermal growth factor (EGF), a 6,700 dalton polypeptide that is abundantly expressed in maternal and fetal tissues, blocks the in vitro TNF/IFN‐induced cytotoxicity of human cytotrophoblasts and syncytiotrophoblasts from normal term placentas. This antagonistic effect is dose‐related (10−10 M EGF, half‐maximal) and proceeds via the interruption of an early step in the cytokine‐induced apoptotic response. These observations suggest a novel role for EGF in normal placental development and indicate that the interplay between EGF, TNFα, and IFNγ may determine the rate of trophoblast growth and renewal during gestation.

In vitro cultured human term cytotrophoblast: A model for normal primary epithelial cells demonstrating a spontaneous differentiation programme that requires EGF for extensive development of syncytium

Normal human term cytotrophoblast cells prepared by trypsin-DNAse I digestion with and without secondary immunological purification with CD9 antibodies were investigated for the expression of morphological and genetic markers of proliferation and differentiation. After 24 h of culture, the cell preparations demonstrated spontaneous formation of microvilli and formation of small syncytial units as assessed by desmoplakin staining and FITC-dextran microinjection. EGF was required for mature syncytial formation. Compared to log-phase proliferating HeLa cells, uptake of [3H]thymidine incorporation was low and quickly decreased to negligible levels. Expression of the proto-oncogenes c-myc, c-fos, and c-jun and histone 2A decreased rapidly in the first 24 h of culture in both cell preparations, followed by an increase in expression of c-fos and junB over the next 3 days of culture. Proto-oncogene changes were similar in attached and suspension cells. Spontaneous increases in alpha hCG, pregnancy-specific beta(1)-glycoprotein and 3 beta-hydroxysteroid dehydrogenase (3 beta OHSD) occurred within 1 day in both cell preparations. EGF receptor blocking antibodies did not inhibit minor degrees of spontaneous syncytial formation nor inhibit spontaneous expression of alpha hCG or 3 beta OHSD mRNA, but did prevent extensive synctialization induced by EGF. The results demonstrate that term cytotrophoblast cells even in serum-free conditions or suspension culture rapidly commit to a non-proliferative differentiation program in culture which includes limited syncytialization and marked hormone mRNA expression. However, EGF is required for extensive syncytial development.

The trophoblast as an integral component of a macrophage-cytokine network

The trophoblast, an epithelial cell of fetal origin that forms the physical barrier between the mother and developing conceptus, becomes a component of the host immune system during pregnancy. Of the classical immune cells, it most closely resembles the macrophage, also present in high numbers in the pregnant uterus. The macrophage and trophoblast, as cell classes, share characteristics such as phagocytosis, syncytialization, invasiveness, expression of the proteins CD4, CD14, IgG receptor (FcR), non‐specific esterase, granulocyte macrophage‐CSF (GM‐CSF), Colony stimulating factor 1 (CSF‐l), interleukin‐I (IL‐1), interleukin‐6 (IL‐6), tumour necrosis factor (TNF‐α), transforming growth factors (TGF), platelet‐α derived growth factor (PDGF) and receptors for these cytokines. In the uterus both cell types appear regulated by a common element, the uterine epithelium, that secretes cytokines such as CSF‐1, GM‐CSF, TNFα, TGFβ, IL‐6, and leukaemia inhibitory factor (LIF) that target both macrophages and trophoblasts. The common characteristics and regulation that make teleological sense in terms of co‐ordinating local uterine immunity during pregnancy may also be important in transmission of congenital diseases such as AIDS. The production by the uterine epithelium of a number of cytokines previously only associated with mononuclear phagocyte production and function predicts the existence of a similar, but broader, shared cytokine network encompassing trophoblast and the principal immune regulatory cell, the T lymphocyte.

Functional, long-term cultures of human term trophoblasts purified by column-elimination of CD9 expressing cells.

We have extended previous observations of expression of the trypsin-resistant cell surface antigen CD9 on placental fibroblasts to virtually all cells in the villous stroma and developed a method for eliminating CD9 expressing cells from trypsinized placental preparations. Preparations incubated with the mouse anti-human CD9 monoclonal antibody 50H.19 were passed through a goat anti-mouse immunoglobulin column that captures CD9 expressing cells. Approximately 95 per cent of the eluted cells stained positive with the villous trophoblast specific antibody GB25 and could be cryopreserved and thawed with > 80 per cent recovery in culture. One week cultures contained fewer than 0.3 per cent vimentin positive (mesenchymal) cells and maintained secretion of hCG. Two week cultures remained free of fibroblasts and macrophages. Clusters of trophoblasts that formed spontaneously during the first week of culture were shown by microinjection of carboxyfluorescein and by staining with anti-desmoplakin antibody to be a patchwork of mononuclear cells and syncytial units. Although the DNA content of the culture decreased by 35 per cent during the 2 week culture, the metabolic capacity and protein content remained relatively constant. Thus, CD9 immuno-elimination gives a high yield of enriched and viable trophoblasts that can be cultured for at least 2 weeks with almost no contamination by stromal cells.

Characterization of cytokine production by the metrial gland and granulated metrial gland cells

The metrial gland and its population of bone marrow-derived, large, granulated, lymphocyte-like cells, termed granulated metrial gland (GMG) cells, are consistent but poorly understood, decidua-associated features of pregnancy in the mouse and other species. Decidua, a complex maternal tissue, is thought to be a source of cytokines important for placental development. Thus, it is important to determine if lymphokine or cytokine production is among the activities of the metrial gland and GMG cells. Media conditioned by culture of either metrial gland explants or migrating GMG cells were evaluated for various cytokine activities. At least four activities were present: CSF-1, IL-1, a factor promoting proliferation of DA-1 cells that was not GM-CSF, IL-3 or erythropoietin and an activity cytotoxic to the CSF-1-dependent macrophage cell line 5/10.14. CSF-1 and IL-1 appeared to be products of the GMG cells. Cytokines not present at detectable levels included IL-2, IL-4, TNF-alpha and TGF-beta. Qualitatively, the cytokine profiles remained constant throughout days 8-16 of gestation. mRNA from migratory GMG cells was isolated and assayed for eleven cytokine mRNAs by polymerase chain reaction-based amplification of cDNA synthesized from mRNA. GMG cell RNA contained transcripts for LIF and CSF-1 but did not contain transcripts for GM-CSF, G-CSF, IL-2, IL-3, IL-4, IL-6, IL-7, IFN-gamma or TNF-alpha. TGF-beta transcripts were detected in occasional samples at very low levels. Since GMG cells are highly mobile cells that migrate throughout the placenta and into trophoblast-lined maternal blood spaces, their function in pregnancy may involve the delivery of very localized differentiation or growth regulatory signals to the developing fetal trophoblast and placenta.

Invasion of the leukocytes into the fetal-maternal interface during pregnancy

No other organ in the body undergoes such an invasion of selective cells (leukocytes) and release of homing molecules, CAMs, proinflammatory cytokines, and mediators or undergoes similar extensive remodeling of tissues over such a short period of time as the pregnant uterus. This is especially interesting, as an infectious process involving microorganisms does not exist in a healthy pregnancy and delivery. Furthermore, after delivery of the baby and placenta, the uterus involutes and returns to its normal monthly cycling, and most of the leukocytes are swept away or leave. In this review, we discuss leukocyte infiltration and recruitment and the potential roles of each subpopulation of leukocytes in relation to pregnancy and the problems of preterm birth, pre‐eclampsia, and intrauterine growth restriction.