The measurement of constitutive intracellular cytokine production in human extravillous cytotrophoblast and decidua by flow cytometry

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Abstracts

4 ng/ml, significantly more monocytes produced intracellular TNF-a in pregnant than non-pregnant women (76.0% versus 64.8% at 40 ng/ml, p < 0.01). Monocytes from normal pregnant women are more sensitive to endotoxin, and the TNF-a response may be more easily elicited in inflammatory conditions in vivo. This methodology enables cytokine response patterns from specific leucocyte subtypes to be studied, and other cytokines including IL-12, IL-4 and interferon-y are presently being investigated. Keywords:

Monocytes;

TNF-a;

Cytokines; Flow cytometry

(721 The measurement of constitutive intracellular cytokine production in human extravillous cytotrophoblast and decidua by flow cytometry I.L. Sargent, L.M. Clover, C.W.G. Redman, G.P. Sacks. NufJield Department of Obstetrics and Gynuecology, John Radcliffe Hospital, Oxford, UK

It has been proposed that in pregnancy secretion of Th2 cytokines by the placenta and decidua causes the downregulation of potentially harmful maternal cell-mediated immune responses. While there is published evidence for the production of IL-4 and IL-10 by these tissues the methods used do not permit simultaneous quantitation of the cytokines and localisation of the cells in which they are produced. We have therefore developed a flow cytometric technique to measure trophoblast intracellular cytokines. Term amniochorion and decidual digests were labelled with antibodies to intracellular cytokines (IL-4, IL-10 and TNFa) by fixing and permeabilising the cells. By combining this in three colour labelling with antibodies to trophoblast (cytokeratin), class I MHC or CD14 it was possible to quantitate the expression of these cytokines in extravillous cytotrophoblast and macrophages in these tissues. The median percentages of cells producing cytokines in term amniochorion and decidua (n = 3) respectively were IL-4 (18% and 4.6%), IL-10 (1.9% and 1.8%) and TNFcl (1.8% and 6.6%). Of the cells producing IL-4, 93% of those in amniochorion were trophoblast (cytokeratin + ve/ CD14 - ve) while in the decidua 83% were macrophages (CD14 + ve). In the decidua 95.5% of the cells producing TNFx were also CD14 + ve. This study has demonstrated the potential of this technique for the identification and quantitation of intracellular cytokines in cytotrophoblast and decidual cells and is being extended to other trophoblast subpopulations at different stages of gestation. Keywords:

cytometry

Thl/Th2

cytokines; IL-4; TNFcr; IL-lo; Cytotrophoblast;

Flow