Molecular evidence for natural killer-like cells in equine endometrial cups.

OBJECTIVES: To identify equine orthologs of major NK cell marker genes and utilize them to determine whether NK cells are present among the dense infiltration of lymphocytes that surround the endometrial cup structures of the horse placenta during early pregnancy. STUDY DESIGN: PCR primers were developed to detect the equine orthologs of NKP46, CD16, CD56, and CD94; gene expression was detected in RNA isolated from lymphocytes using standard 2-step reverse transcriptase (RT) PCR and products were cloned and sequenced. Absolute real-time RT-PCR was used to quantitate gene expression in total, CD3+, and CD3- peripheral lymphocytes, and invasive trophoblast. Lymphocytes surrounding the endometrial cups (ECL) of five mares in early pregnancy were isolated and NK marker gene expression levels were assayed by quantitative RT-PCR. MAIN OUTCOME MEASURES: Absolute mRNA transcript numbers were determined by performing quantitative RT-PCR and comparing values to plasmid standards of known quantities. RESULTS: NKP46 gene expression in peripheral CD3- lymphocytes was higher than in CD3+ lymphocytes, CD16 levels were higher in the CD3+ population, and no significant differences were detected for CD56 and CD94 between the two groups. Expression of all four NK cell markers was significantly higher in lymphocytes isolated from the endometrial cups of pregnant mares compared to PBMC isolated from the same animal on the same day (NKP46, 14-fold higher; CD94, 8-fold higher; CD16, 20-fold higher; CD56, 44-fold higher). CONCLUSIONS: These data provide the first evidence for the expression of major NK cell markers by horse cells and an enrichment of NK-like cells in the equine endometrium during pregnancy.

Modeling trophoblast differentiation using equine chorionic girdle vesicles.

The chorionic girdle of the equine conceptus is comprised of specialized trophoblast cells which, at day 36-38 of equine pregnancy, gain an invasive phenotype and invade the endometrium to form endometrial cups. Studies of equine endometrial cups remain difficult to perform because of the invasive techniques required to obtain cup tissue and because sampling requires termination of the pregnancy. In this study we developed a system to model trophoblast differentiation and trophoblast-immune interactions in vitro and in vivo. We utilized a method of culturing chorionic girdle pieces in serum-free medium to promote spontaneous formation of vesicle structures enriched for terminally differentiated binucleate cells that secreted equine chorionic gonadotrophin (eCG). Immunohistochemical staining and scanning electron microscopy showed that the cells of the vesicles closely resembled the outer layers of chorionic girdle immediately prior to invasion. Chorionic girdle vesicles were harvested after 72h in culture and ectopically transplanted via injection into the vulvar mucosa of recipient mares. At 7, 14, 21 and 28days after transplantation, biopsies of the injection sites were obtained. Immunohistochemical labeling of cryostat sections of the biopsies with a panel of monoclonal antibodies to horse trophoblast molecules demonstrated survival, differentiation, and presence of trophoblast cells for at least 21days. Serial sections of the biopsies labeled with antibodies to the equine lymphocyte surface markers CD4 and CD8, together with lymphocyte microcytotoxicity assays, revealed that the recipients mounted both cellular and humoral antibody immune responses to the transplanted trophoblast cells. This new method for culturing equine chorionic girdle trophoblast cells, and for transplanting trophoblast vesicles to ectopic sites, should allow identification of key aspects of trophoblast differentiation and the interactions that occur between invasive trophoblast and the maternal immune system.