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Functional expression of CSF-1 receptors on normal human trophoblast
.4bstracts: European Placenta Group
monocyte/macrophages. The lack of functional maturation may explain the rarity of chronic villitis early in gestation.
199
of the villous stromal macrophage
FIBRIN MATRIX MODULATES THE PHENOTYPIC DIFFERENTIATION AND PROLIFERATION OF HUMAN PLACENTAL TROPHOBLAST D. M. Nelson, E. C. Crouch, E. C. Curran 8z D. R. Farmer (Department of Obstetrics and Gynaecology, and Pathology, Jewish Hospital of St. Louis, Washington University School of Medicine, St. Louis, MO, USA) We used immunocytochemistry, morphometric analysis, and electron microscopy to study perivillous fibrin deposits on normal term human placental villi, and we examined the response of cultured cellular trophoblast to fibrin matrix in vitro. Histologically, the perivillous deposits were hypocellular, eosinophilic masses immunoreactive for the B-/l monomer of fibrin II. Of 3477 villi examined, 258 villous profiles had denudations of the syncytial trophoblast and 191 (74.4 per cent) of these, or 5.5 per cent of the total villi, had associated fibrin deposits. Ultrastructurally, damage to the syncytial trophoblast was apparent at the edge of some deposits, where syncytial denudation was accompanied by a fibrin coating of residual cellular trophoblast and trophoblastic basal lamina. Other deposits were surfaced by syncytial trophoblast with underlying cellular trophoblast and new basal lamina external to the basal lamina of the villous core. Cultured cellular trophoblast grown on a fibrin matrix, but not on uncoated plastic, differentiated cytologically and histologically into a trophoblast layer like that on term villi. Trophoblast cultures grown 72 h on fibrin had medium levels of HCG (225 mIU/ml), oestradiol(151 I pg/ml) and progesterone (45 ng/ml) comparable to cultures grown without matrix. However, the labelling index of cells grown 24 h on fibrin (0.49 per cent f 0.57 per cent) was rz-fold lower than cells grown on plastic without matrix (5.82 per cent f 1.85 per cent). We suggest that re-epithelialization of perivillous fibrin deposits is a form of villous repair and that trophoblast-fibrin interactions modulate trophoblastic differentiation and proliferation. (Supported by NICHHD Grant 22913 to DMN).
FUNCTIONAL EXPRESSION OF CSF-I RECEPTORS ON NORMAL HUMAN TROPHOBLAST M. Garcia-Lloret, L. Guilbert & D. W. Morrish (Departments of Immunology and Medicine, University of Alberta, Edmonton, Alberta, Canada T6G 2G3) CSF-I is a haematopoietic growth factor originally identified by its ability to stimulate the proliferation, differentiation, and survival of cells of the monocyte-macrophage lineage, effects mediated by binding to a single class of high affinity receptors, product of the c-fms protooncogene. Recent reports suggest an important role of CSF-r in normal pregnancy. CSF-I increases ~ooo-fold in uterine tissues during murine pregnancy and expression of c-fms RNA has been demonstrated in choriocarcinoma cell lines and human placenta. We have previously shown that choriocarcinoma cell lines (BeWo, JAR, JEG-3) have CSF-I receptors, respond to this factor, and are able to secrete it. Thus, we investigated whether these features are also a characteristic of normal human trophoblast. Purified term villous trophoblasts (over 95 per cent cytokeratin positive, vimentin negative, macrophage marker negative) expressed c-fms mRNA and bound 125-I-CSF-I in a specific and saturable manner. In situ hybridization of first trimester placenta indicated the presence of c-fms RNA in syncytiotrophoblast, located in
500
Placenta (1989), Vol. IO
the rim of the chorionic villi. No mitogenic response was obtained when pure trophoblasts were stimulated with recombinant human CSF-I. However, production of human placental lactogen increased by two fold when the cells were cultured in the presence of this factor. Low levels of endogenous CSF-I production were transiently detected in culture supernatant using a RRA. Our results indicate that CSF-I may have a regulatory role in normal trophoblast differentiated function.
VARIABLE EPIDERMAL GROWTH FACTOR-GROWTH FACTOR RECEPTOR REGULATION AMONG NORMAL TROPHOBLAST, FIBROBLASTS, AND TUMOR CELLS D. W. Morrish, R. Sasi, C. C. Lin, D. Bhardwaj 8z S. Shiferaw (Departments of Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada T6G 2G3) Epidermal growth factor (EGF) in d uces proliferation of fibroblasts, some tumour cells, and also induces differentiation of normal human trophoblast (Morrish et al, 1987,~ournal of Clinical Endocrinology and Metabolism, 65, 1282). Because both up- and down-regulation of EGF receptors (EGF-R) have been reported, and because we wished to determine the molecular events during proliferation and differentiation of cells, we studied EGF effects on its receptor in two breast cancer cell lines (AA,LW), A431 cells (epidermoid carcinoma), normal human term placental fibroblasts, and normal isolated term human trophoblast. AA and LW cells were grown from biopsies. Trophoblast was prepared as described (above reference) and fibroblasts were grown from crude placental preparations. All cell types were cultured for 3 days prior to testing in serum-free medium (DMEM or Ham’s FIO), then exposed for 3 and 24 h to 1ong/m1 EGF. Controls received BSA. Cells were removed after ECF exposure and RNA extracted with guanidinium isothiocyanate and CSCI gradient centrifugation. RNA was slot blotted to nylon membranes and hybridized with the human EGF receptor cDNA probe. Results showed no change after 3 h EGF exposure in any cell type except a slight increase in AA cells. After 24 h of EGF exposure, EGF-R mRNA expression did not change in AA, LW, A431 or fibroblast cells. However, trophoblast demonstrated a 20-50 per cent decrease in EGF-R mRNA compared to controls. We thus conclude that normal trophoblast downregulates its EGF-R mRNA, but that normal placental fibroblasts and three tumour cell lines do not. Trophoblast in this culture system is non-proliferative compared to the other cells used. Therefore, differences in EGF-R response to EGF among trophoblasts, fibroblasts, and tumour cells may be useful in determining controlling mechanisms of normal and neoplastic cell growth. (Supported by the Medical Research Council of Canada.)
SYNTHESIS OF EIGHT SERUM PROTEINS BY GLANDULAR EPITHELIAL CELLS OF GESTATIONAL ENDOMETRIUM T. N. Fay”, B. Teisneti &J. G. Grudzinskas” (” Academic Unit, Department of Obstetrics and Gynaecology, The London Hospital, Whitechapel, London EI IBB, UK and bInstitute of Medical Microbiology, Odense University, Odense, Denmark 5000) Human uterine luminal fluid is known to contain serum proteins but the origin of these proteins remains unclear. The aim of this study was to identify the synthesis of specific serum proteins by gestational endometrial (GE) glandular epithelium. GE was fractioned by enzyme digestion and monolayer cultures of glandular epithelium
monocyte/macrophages. The lack of functional maturation may explain the rarity of chronic villitis early in gestation.
199
of the villous stromal macrophage
FIBRIN MATRIX MODULATES THE PHENOTYPIC DIFFERENTIATION AND PROLIFERATION OF HUMAN PLACENTAL TROPHOBLAST D. M. Nelson, E. C. Crouch, E. C. Curran 8z D. R. Farmer (Department of Obstetrics and Gynaecology, and Pathology, Jewish Hospital of St. Louis, Washington University School of Medicine, St. Louis, MO, USA) We used immunocytochemistry, morphometric analysis, and electron microscopy to study perivillous fibrin deposits on normal term human placental villi, and we examined the response of cultured cellular trophoblast to fibrin matrix in vitro. Histologically, the perivillous deposits were hypocellular, eosinophilic masses immunoreactive for the B-/l monomer of fibrin II. Of 3477 villi examined, 258 villous profiles had denudations of the syncytial trophoblast and 191 (74.4 per cent) of these, or 5.5 per cent of the total villi, had associated fibrin deposits. Ultrastructurally, damage to the syncytial trophoblast was apparent at the edge of some deposits, where syncytial denudation was accompanied by a fibrin coating of residual cellular trophoblast and trophoblastic basal lamina. Other deposits were surfaced by syncytial trophoblast with underlying cellular trophoblast and new basal lamina external to the basal lamina of the villous core. Cultured cellular trophoblast grown on a fibrin matrix, but not on uncoated plastic, differentiated cytologically and histologically into a trophoblast layer like that on term villi. Trophoblast cultures grown 72 h on fibrin had medium levels of HCG (225 mIU/ml), oestradiol(151 I pg/ml) and progesterone (45 ng/ml) comparable to cultures grown without matrix. However, the labelling index of cells grown 24 h on fibrin (0.49 per cent f 0.57 per cent) was rz-fold lower than cells grown on plastic without matrix (5.82 per cent f 1.85 per cent). We suggest that re-epithelialization of perivillous fibrin deposits is a form of villous repair and that trophoblast-fibrin interactions modulate trophoblastic differentiation and proliferation. (Supported by NICHHD Grant 22913 to DMN).
FUNCTIONAL EXPRESSION OF CSF-I RECEPTORS ON NORMAL HUMAN TROPHOBLAST M. Garcia-Lloret, L. Guilbert & D. W. Morrish (Departments of Immunology and Medicine, University of Alberta, Edmonton, Alberta, Canada T6G 2G3) CSF-I is a haematopoietic growth factor originally identified by its ability to stimulate the proliferation, differentiation, and survival of cells of the monocyte-macrophage lineage, effects mediated by binding to a single class of high affinity receptors, product of the c-fms protooncogene. Recent reports suggest an important role of CSF-r in normal pregnancy. CSF-I increases ~ooo-fold in uterine tissues during murine pregnancy and expression of c-fms RNA has been demonstrated in choriocarcinoma cell lines and human placenta. We have previously shown that choriocarcinoma cell lines (BeWo, JAR, JEG-3) have CSF-I receptors, respond to this factor, and are able to secrete it. Thus, we investigated whether these features are also a characteristic of normal human trophoblast. Purified term villous trophoblasts (over 95 per cent cytokeratin positive, vimentin negative, macrophage marker negative) expressed c-fms mRNA and bound 125-I-CSF-I in a specific and saturable manner. In situ hybridization of first trimester placenta indicated the presence of c-fms RNA in syncytiotrophoblast, located in
500
Placenta (1989), Vol. IO
the rim of the chorionic villi. No mitogenic response was obtained when pure trophoblasts were stimulated with recombinant human CSF-I. However, production of human placental lactogen increased by two fold when the cells were cultured in the presence of this factor. Low levels of endogenous CSF-I production were transiently detected in culture supernatant using a RRA. Our results indicate that CSF-I may have a regulatory role in normal trophoblast differentiated function.
VARIABLE EPIDERMAL GROWTH FACTOR-GROWTH FACTOR RECEPTOR REGULATION AMONG NORMAL TROPHOBLAST, FIBROBLASTS, AND TUMOR CELLS D. W. Morrish, R. Sasi, C. C. Lin, D. Bhardwaj 8z S. Shiferaw (Departments of Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada T6G 2G3) Epidermal growth factor (EGF) in d uces proliferation of fibroblasts, some tumour cells, and also induces differentiation of normal human trophoblast (Morrish et al, 1987,~ournal of Clinical Endocrinology and Metabolism, 65, 1282). Because both up- and down-regulation of EGF receptors (EGF-R) have been reported, and because we wished to determine the molecular events during proliferation and differentiation of cells, we studied EGF effects on its receptor in two breast cancer cell lines (AA,LW), A431 cells (epidermoid carcinoma), normal human term placental fibroblasts, and normal isolated term human trophoblast. AA and LW cells were grown from biopsies. Trophoblast was prepared as described (above reference) and fibroblasts were grown from crude placental preparations. All cell types were cultured for 3 days prior to testing in serum-free medium (DMEM or Ham’s FIO), then exposed for 3 and 24 h to 1ong/m1 EGF. Controls received BSA. Cells were removed after ECF exposure and RNA extracted with guanidinium isothiocyanate and CSCI gradient centrifugation. RNA was slot blotted to nylon membranes and hybridized with the human EGF receptor cDNA probe. Results showed no change after 3 h EGF exposure in any cell type except a slight increase in AA cells. After 24 h of EGF exposure, EGF-R mRNA expression did not change in AA, LW, A431 or fibroblast cells. However, trophoblast demonstrated a 20-50 per cent decrease in EGF-R mRNA compared to controls. We thus conclude that normal trophoblast downregulates its EGF-R mRNA, but that normal placental fibroblasts and three tumour cell lines do not. Trophoblast in this culture system is non-proliferative compared to the other cells used. Therefore, differences in EGF-R response to EGF among trophoblasts, fibroblasts, and tumour cells may be useful in determining controlling mechanisms of normal and neoplastic cell growth. (Supported by the Medical Research Council of Canada.)
SYNTHESIS OF EIGHT SERUM PROTEINS BY GLANDULAR EPITHELIAL CELLS OF GESTATIONAL ENDOMETRIUM T. N. Fay”, B. Teisneti &J. G. Grudzinskas” (” Academic Unit, Department of Obstetrics and Gynaecology, The London Hospital, Whitechapel, London EI IBB, UK and bInstitute of Medical Microbiology, Odense University, Odense, Denmark 5000) Human uterine luminal fluid is known to contain serum proteins but the origin of these proteins remains unclear. The aim of this study was to identify the synthesis of specific serum proteins by gestational endometrial (GE) glandular epithelium. GE was fractioned by enzyme digestion and monolayer cultures of glandular epithelium