M. Garcia-Lloret

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.

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.

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.

Analysis of the synergistic stimulation of mouse macrophage proliferation by macrophage colony-stimulating factor (CSF-1) and tumor necrosis factor alpha (TNF-alpha).

Abstract: Tumor necrosis factor a (TNF‐α) more than doubles tritiated thymidine ([3H]TdR) uptake in mouse macrophages stimulated by macrophage colony‐ stimulating factor (CSF‐1). However, nothing is known of how TNF‐α affects this increase or even whether it is manifested by increased cellular proliferation. Here we characterize the effects of TNF‐α on CSF‐l‐stimulated proliferation of both primary cells (bone marrow‐derived macrophages, BMMs) and a cloned growth factor‐dependent macrophage cell line (SI). We show that the TNF‐ α‐induced increase in [3H]TdR uptake of CSF‐l‐stimu‐ lated macrophages is directly proportional to an increase in the DNA content of the culture and that the effects of TNF‐α are direct and independent of cell number. TNF‐a decreases the population doubling time of log‐phase growing macrophages having quite different growth rates to the same (approximately 30%) extent: the doubling time of BMMs decreases from 24 to 17 h and that of SI cells from 17 to 13 h. TNF‐α exerts its effects on log‐phase growth by increasing to the same proportion CSF‐l‐stimulated proliferation at all concentrations of CSF‐1; that is, TNF‐α does not shift, but rather amplifies, the CSF‐1 dose‐response curve. Although TNF‐α alone does not stimulate macrophage proliferation, its presence in S1 cell cultures coming to quiescence after withdrawal of CSF‐1 greatly increases subsequent CSF‐l‐stimulated [3H]TdR uptake as the cells reenter the cycle. Finally, we show that both human and mouse TNF‐α increase CSF‐l‐stimulated log‐phase growth and reentry of quiescent cells into the cycle equally on a molar basis (halfmaximal stimulation of approximately 0.3 nM). The latter observation argues that the growth‐stimulatory effects of TNF‐α are mediated via the 55‐60‐kd TNF receptor. We conclude that TNF‐α acts directly on growth‐ competent macrophages to decrease significantly the population doubling time in a manner that enhances the mitogenic effects of CSF‐1.

Tilt Toward TH2 in Successful Pregnancy

The hypothesis explored in this chapter is that the immune system of the pregnant female is shifted toward antibody-mediated immunity regulated by TH2 helper cells as a result of spontaneous cytokine production by placental-decidual tissues. This alteration of cytokine balance in turn has pronounced consequences for maternal health and fetal survival. We have recently reviewed clinical and experimental evidence in support of this point of view (1) and briefly summarize it here.