Nitric Oxide Synthase-2 and Expression of Perforin in Uterine NK Cells

In human, mouse, and rat pregnancy, maternal NK cells accumulate and differentiate at implantation sites. These cells, termed uterine NK (uNK) cells, express NO synthase (NOS)-2 and develop cytolytic molecules such as perforin and granzymes during differentiation in situ. In this study, relationships between expression of the NOS-2 gene, uNK cell population density and tissue distribution, and synthesis of perforin were investigated. Uteri from wild-type (WT) and NOS-2−/− mice were collected at gestation days (g.d.) 8, 10, 12, 14, and 16 (n, >2/g.d.). Histochemical staining failed to reveal any differences between the population densities or tissue distributions of uNK cells in WT and NOS-2−/− uteri at any stage of gestation. By contrast, immunohistochemical staining with anti-perforin Abs demonstrated significantly fewer perforin-positive uNK cells in two uterine compartments of NOS-2−/− mice in comparison to the same compartments in WT mouse uteri. Perforin-positive uNK cells were reduced in NOS-2−/− metrial glands at g.d. 8, 10, and 12 and in decidua basalis at g.d. 12 (p < 0.05). Analysis of perforin protein by immunoblotting confirmed this observation. Northern blot hybridization studies showed that loss of perforin protein in NOS-2−/− mice was accompanied by decreased steady-state levels of perforin mRNA. These results demonstrate that migration of uNK cells into the uterus, selection of residency sites, and proliferation in situ are independent of NOS-2. By contrast, their differentiation, including transcription and translation of the cytotoxic molecule perforin, was shown to rely on normal expression of the NOS-2 gene.