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Human trophoblast culture supernatants inhibit a mouse PLN assay both in primary and secondary responses
P 221
155
I N T R A C E L L U L A R CALCIUM LEVELS IN LYMPHOCYTES UNDER CONDITIONS OF
ROSETTE I N H I B I T I O M TEST FOR THE DETECTION CF EARLY PREGNANCY FACTOR HANF,V., OETTLING, G., ADAMSON, L., TINNEBERG, H.-R. U n i v e r s i t y WomenJ s Hospital of the E b e r h a r d - K a r l s - U n i v e r s i t ~ t T,",bingen, S c h l e i c h s t r . q . , O-7q00 TE~bingne, West-Germany Much controversy has been raised about the very existence of Early Pregnancy Factor (EPF) and the only method of its detection so far, the Rosette Inhibition Test. As this admittedly d i f f i c u l t test system works satisfyingly in our hands, i t was the objective of this study to demonstrate another reproducible and objectively measurable effect of EPF treatment in lymphocytes. As a source of EPF we used a concentrate from urine of pregnant women which displayed very high rosette inhibiting (i.e. EPF-) a c t i v i t y in the Rosette Inhibition Test. The free cytoplasmic Ca ++ concentration of freshly prepared human ]ymphocytes was measured using the calcium sensitive flourescent dye Fura-2, which was trapped in the cells by preincubation in the presence of the membranepermeant ester derivative Fura-2-acetoxymethyle~;ter for 30 rain. at 37°C. Fluorescence intensities of the labelled cell suspensions (5 x l 0 cells/ml) were measured at 37°C in a fluorometer cuvette equipped with a magnetic stirrer (wavelengths: 350 nm for excitation, 500 nm for emission). The cells were successively treated with the various components used in the Rosette Inhibition Test in their appropriate order EPF-concent r a t e or equivalent fraction from non-pregnant urine as control, guinea pig complement, anti-CD-2-antibody and sheep red blood cells. In neither of the various steps was a significant change in the cytoplasmic Ca ++ levels noticed. Controls using Concanavalin-A as a known intracellular calcium liberator elicited prompt changes in the free cytoplasmic Ca ++ levels. Summary: Under the experimental conditions chosen i t was not possible to demonstrate any effect of EPF on the intracellular calcium homeostasis of lymphocytes in the set up of the Rosette Inhibition Test.
I(eyworGs: Early Pregnancy Factor, Calcium, Fura-2 P 222 HUMAN TROPHOBLAST CULTURE SUPERNATANTS INHIBIT A MOUSE PLN ASSAY BOTH IN PRIY~ARY AND SECONDARY RESPONSES. Elisabeth MENU, R. KINSKY and G. CHAOUAT I N S E R M U 262, Clinique Baudelocque, 123 Bld. de Port-Royal,
75674-Paris
Human trophoblast culture supernatants were shown in previous studies to inhibit in vitro responses of human and murine lymphooytes reacting in an MLR-CML system and to modulate proliferation of an Ii-2 dependent cell line or a cytotoxicity of cloned human T cells. In the present work use was made of an in vivo assay, the popliteal lymph node (PLN) test. In the primary reaction, culture supernatants were added directly to the allogenei~ spleen cells injected into the foot pads of recipient mice and PLNs were harvested 4 to 6 days later. In the secondary response, the supernatants were mixed with allogeneic immunizing cells injected i.p. to the prospective cell donors and their spleens used 7 days later for the induction of secondary responses in the recipient's foot pads. PLNs were harvested 3 days later. The results show an inhibition of both types of responses evaluated simultaneously by the respective PLN weights as well as by the proliferative response measured by 3H incorporation. Controls, consisting of mice receiving cells treated with o±h~r non-placental xenogeneio proteins, have not shown any inhibition of the response intensity obtained by cells injected in medium alone. Our data suggest a potential use of human trophoblast supernatants in the monitoring of transplantation reactions and are adding further emphasis on the local immunomodulatory role of placental glycoproteins in even xenogeneic types of gestation. Keywords: Human trophoblast supernatantg, PLN-Assay inhibition.
155
I N T R A C E L L U L A R CALCIUM LEVELS IN LYMPHOCYTES UNDER CONDITIONS OF
ROSETTE I N H I B I T I O M TEST FOR THE DETECTION CF EARLY PREGNANCY FACTOR HANF,V., OETTLING, G., ADAMSON, L., TINNEBERG, H.-R. U n i v e r s i t y WomenJ s Hospital of the E b e r h a r d - K a r l s - U n i v e r s i t ~ t T,",bingen, S c h l e i c h s t r . q . , O-7q00 TE~bingne, West-Germany Much controversy has been raised about the very existence of Early Pregnancy Factor (EPF) and the only method of its detection so far, the Rosette Inhibition Test. As this admittedly d i f f i c u l t test system works satisfyingly in our hands, i t was the objective of this study to demonstrate another reproducible and objectively measurable effect of EPF treatment in lymphocytes. As a source of EPF we used a concentrate from urine of pregnant women which displayed very high rosette inhibiting (i.e. EPF-) a c t i v i t y in the Rosette Inhibition Test. The free cytoplasmic Ca ++ concentration of freshly prepared human ]ymphocytes was measured using the calcium sensitive flourescent dye Fura-2, which was trapped in the cells by preincubation in the presence of the membranepermeant ester derivative Fura-2-acetoxymethyle~;ter for 30 rain. at 37°C. Fluorescence intensities of the labelled cell suspensions (5 x l 0 cells/ml) were measured at 37°C in a fluorometer cuvette equipped with a magnetic stirrer (wavelengths: 350 nm for excitation, 500 nm for emission). The cells were successively treated with the various components used in the Rosette Inhibition Test in their appropriate order EPF-concent r a t e or equivalent fraction from non-pregnant urine as control, guinea pig complement, anti-CD-2-antibody and sheep red blood cells. In neither of the various steps was a significant change in the cytoplasmic Ca ++ levels noticed. Controls using Concanavalin-A as a known intracellular calcium liberator elicited prompt changes in the free cytoplasmic Ca ++ levels. Summary: Under the experimental conditions chosen i t was not possible to demonstrate any effect of EPF on the intracellular calcium homeostasis of lymphocytes in the set up of the Rosette Inhibition Test.
I(eyworGs: Early Pregnancy Factor, Calcium, Fura-2 P 222 HUMAN TROPHOBLAST CULTURE SUPERNATANTS INHIBIT A MOUSE PLN ASSAY BOTH IN PRIY~ARY AND SECONDARY RESPONSES. Elisabeth MENU, R. KINSKY and G. CHAOUAT I N S E R M U 262, Clinique Baudelocque, 123 Bld. de Port-Royal,
75674-Paris
Human trophoblast culture supernatants were shown in previous studies to inhibit in vitro responses of human and murine lymphooytes reacting in an MLR-CML system and to modulate proliferation of an Ii-2 dependent cell line or a cytotoxicity of cloned human T cells. In the present work use was made of an in vivo assay, the popliteal lymph node (PLN) test. In the primary reaction, culture supernatants were added directly to the allogenei~ spleen cells injected into the foot pads of recipient mice and PLNs were harvested 4 to 6 days later. In the secondary response, the supernatants were mixed with allogeneic immunizing cells injected i.p. to the prospective cell donors and their spleens used 7 days later for the induction of secondary responses in the recipient's foot pads. PLNs were harvested 3 days later. The results show an inhibition of both types of responses evaluated simultaneously by the respective PLN weights as well as by the proliferative response measured by 3H incorporation. Controls, consisting of mice receiving cells treated with o±h~r non-placental xenogeneio proteins, have not shown any inhibition of the response intensity obtained by cells injected in medium alone. Our data suggest a potential use of human trophoblast supernatants in the monitoring of transplantation reactions and are adding further emphasis on the local immunomodulatory role of placental glycoproteins in even xenogeneic types of gestation. Keywords: Human trophoblast supernatantg, PLN-Assay inhibition.