The final digestate was passed through a 23G needle to dissociate the remaining cell clusters. The isolated cells were centrifuged at 1500 rpm for 5 min at 4°C, and washed in Ham F-10 medium. This procedure was repeated three times and the final cell pellet was resuspended (1 g of tissue/ml) in 20% Percoll (Sigma), layered on a 60%:50%:40% discontinuous Percoll gradient, and then centrifuged at 22000 rpm for 20 min at 4°C. The top cell layer was collected, washed, resuspended in Ham F-10, and centrifuged at 1500 rpm for 5 min at 4°C.
For MIF staining, sections (4-im thickness) of paraffin-embedded placenta tissues were cut, deparaffinized, rehydrated, and washed in Tris-buffered saline (TBS; 20 mM Tris-HCl [pH 7.6], 150 mM NaCl). TBS was used for all subsequent washes and for dilution of the antibody. Antigen retrieval was carried out by incubating sections in 10 mM sodium citrate buffer (pH 6.0) in a microwave oven at 750 W for 5 min. For CSF1 immunostaining, frozen tissues were cut (6-im thickness) and fixed in ice-cold acetone.
Anti-human MIF and CSF1 goat polyclonal antibodies were purchased from R&D Systems (Abingdon, UK) and Santa Cruz Biotechnology (Santa Cruz, CA), respectively. Horseradish peroxidase-conjugated rabbit anti-goat antibody was obtained from Calbiochem (San Diego, CA). All the chemicals were of analytical grade (Sigma Chemical Co., St. Louis, MO).
In keeping with its known immunomodulatory functions, we have proposed the involvement of MIF in regulating macrophage accumulation in the pregnant endometrium.
The current study evaluated the involvement of CSF1 and MIF in the recruitment and maintenance of macrophages in human decidua. After demonstrating the presence of these two potentially antagonistic chemokines by immunohistochemical staining of sections of term decidua, we sought to elucidate the mechanisms underlying decidual CSF1 and MIF expression.
These mice are depleted of circulating monocytes and tissue macrophages in several organs, including the uterus. They experience severely reduced fertility, lower implantation rates, and greater embryonic wastage compared with wild-type females. Paradoxically, excess decidual macrophage infiltration has been linked to preeclampsia and intrauterine growth restriction in humans.
The early pregnant human endometrium (decidua) contains a diverse population of leukocytes that mediate innate immunity, including uterine natural killer (uNK) cells (7075%) and macrophages (20-25%). In contrast, T and B lymphocytes, which mediate adaptive immunity, account for 10% and between 1% and 3 % of the white cell population, respectively. In recent years, research has focused on the role played by uNK in placentation, with far less attention being directed towards decidual macrophages.