Blood. in mediating the biological effects of prostaglandins. By utilizing selective antagonists of individual receptors, we have established that this immunosuppressive effect of PGE2 on CTLL-2 cells is usually exerted via the EP4 receptor. Thus, addition of an EP4-selective antagonist, but not of EP1 or EP3 antagonists, abolished the immunosuppressive effect of PGE2 on CTLL-2 cells. This may have implications for attempts to selectively manipulate T-cell responses. host reactions in mice [1,27C29]. A major active component of the immunosuppressive activity was an undefined heat-stable molecule of less than 3 kDa. Chaouat proposed that this factor could, Radicicol under appropriate conditions, bind to proteins produced by the placenta or elsewhere, thereby explaining the fact that molecules such as human chorionic gonadotrophin and -fetoprotein have Radicicol been reported as having immunosuppressive properties which disappear when the protein is usually highly purified or used in recombinant form . We have investigated the immunosuppressive material produced by the chorionic villi of term placenta and report here that this factor suppressing the IL-2-dependent proliferation of CTLL-2 cells is usually PGE2. This function is usually exerted through the EP4 family of receptors. MATERIALS AND METHODS Preparation of human placental supernatants Human placental supernatants (HPS) were obtained as described by Menu . Briefly, human placentas were obtained at term from caesarean deliveries. Chorionic villi were isolated from surrounding tissue and further cut with scissors to obtain 1C3-mm3 pieces. The fragments were washed five times in serum free RPMI-1640 culture medium (Gibco BRL, Paisley, UK). Ex-plants of chorionic villi were cultured at 37C in 5% CO2, using 25 cm3 tissue culture flasks with 20C30 fragments per 10C15 ml serum-free RPMI-1640 supplemented with Radicicol 100 Units/ml penicillin and 100 Radicicol g/ml streptomycin (Gibco GPX1 BRL) and 25 m 2-mercaptoethanol. Supernatants were collected after 48 h, centrifuged for 20 min at 30 000 g at 4C to remove particulate material and stored in aliquots at C80C until use. To obtain low molecular weight material supernatants were ultrafiltered using a Centriprep-3 centrifugal filter unit (Millipore, Watford, UK) with a 3-kDa cut-off. HPS obtained from different placentas are designated using numbers (HPS1-HPS17) and the numbering of the low molecular weight fitrates correspond to the HPS from which they were obtained. In some experiments the filtrate was heated at 100C for 2h prior to use. In experiments investigating the effect of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) inhibition, the chorionic villi explants were prepared as above and after the cleaning procedure ahead of culture had been incubated for 1 h in the current presence of indomethacin (2 g/ml), diclofenac (3 g/ml) (both from Sigma, Poole, UK) or the COX-2-selective inhibitor DFP 3-(2-propyloxy)-(4-methyl-sulphonylphenyl)-(5,5-dimethyl)-furanone] (1 g/ml) (something special from Merck-Frosst, Canada). The explants had been then washed once again and cultured in refreshing medium supplemented using the same levels of indomethacin, dFP or diclofenac. Pursuing 48h of culture the supernatants had been prepared and harvested as over. Immunoregulatory activity of placental supernatants Placental supernatants had been tested for his or her immunosuppressive activity using an IL-2-reliant CTLL-2 cell proliferation assay. CTLL-2 cells had been expanded in RPMI-1640 including 2 mm l-glutamine (Gibco BRL), 100 Devices/ml penicillin and 100 g/ml streptomycin, 25 m 2-mercaptoethanol, 10% heat-inactivated fetal leg serum (HIFCS) and 10 ng/ml recombinant human being interleukin-2 (rhIL-2) (Peprotech Inc, Rocky Hill, NJ, USA). To use Prior, the CTLL-2 cells had been washed in revised Eagles moderate (MEM, Gibco BRL) including 2% HIFCS and incubated for 2C3 h without IL-2. The cells had been after that cultured (5 104 cells/ml) in 96-well flat-bottomed microtitre plates in your final level of 100 l with your final focus of 06 ng/ml of rhIL-2 and in the current presence of placental supernatant or filtrate. Pursuing incubation for 24 h, their proliferation was established utilizing a 4-h pulse of 20 l per well of 5 mg/ml MTT (3-4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) (Sigma) in PBS, or 1 Ci per well 3H]-thymidine (Amersham Pharmacia Biotech). The MTT reactions had been stopped with the addition of 100 l per well of 10% sodium dodecyl sulphate in 001m HCl, and after an over night incubation the quantity of formazan created was measured utilizing a dual wavelength (570 and 630 nm) microtitre dish reader. In a few tests CTLL-2 cells had been preincubated using the PGE2 receptor antagonists L-818 638, L-826 266 and/or L-161 982  (each at 1 m/ml, something special from Merck-Frosst, Canada) for 15 h before the addition of either PGE2 or the placental supernatant.