Implication of Interferon Stimulated Gene 15 (ISG15) in the Recruitment of Uterine Natural Killer Cells into the Murine Implantation Site.

Abstract

Interferon stimulated gene 15 (ISG15) encodes a ubiquitin-like protein that has being implicated in a number of physiological events including innate immune response, interferon signaling and cancer. ISG15 also is induced by pregnancy in the endometrium of rodents, primates and ruminants. Previous reports from our laboratory indicate that expression of ISG15 in the uterus is functionally required for maintenance of pregnancy in mice. Isg15 null females (Isg15-/-) exhibit embryo mortality ranging from 30-70% by 12.5 days post coitum (dpc), in contrast to 4% embryo mortality observed in wild type control females (Isg15+/+). As a consequence, Isg15+/+ mice have larger litters (7.94 ± 0.32 pups/ litter) when compared to Isg15-/- mice (4.2 ± 0.24). Additionally, Isg15-/- females carrying heterozygous embryos showed similar embryonic mortality rates when compared to Isg15-/- females carrying Isg15-/- embryos. This finding suggests that maternal Isg15 genotype contributes to embryonic death. Uterine natural killer cells (uNK) are the most prevalent maternal uterine immune cell and they are found in large number at the feto-maternal interface in mice. They interact closely with the invading placental trophoblast cells, which are responsible for remodeling of spiral arteries. This process is essential to ensure a normal blood supply to the fetus and placenta throughout pregnancy. Uteirne NK cells also produce cytokines (interferon gamma and interleukins 18 and 27), and proliferate within the myometrium at each implantation site forming the mesometrial lymphoid aggregate of pregnancy (MLAp). Uterine NK cells have been linked with failure of pregnancy: studies in mice lacking uNK cells and components of IFN-gamma pathway indicate that uNK cell-derived IFN-gamma is essential for spiral artery remodeling. Because Isg15 is a component of the IFN pathway, we hypothesized that uNK population may be altered in Isg15-/-. To address this question we designed a set of experiments to compare the number and distribution of uNK cells at implantation sites between Isg15+/+ and Isg15-/- mice. Implantation sites of 7.5 dpc and 12.5 dpc (n=5 each genotype and day) were fixed in 4% parformaldehyde and embedded in paraffin. Five μm sections were cut from paraffin-embedded tissues and uNK cells were identified using Dolichos biflorus agglutinin (DBA) lectin histochemistry. The number of uNK was assessed through a computer-assisted image analysis (Image Pro Plus for Windows, Ver 4.0, Media Cybernetics, Silver Spring, MD) at 200 X. A minimum of 3 non-adjacent mid-sagittal sections 40 μm apart were analyzed per implantation site. There was a very conspicuous difference in the density and distribution of uNK cells between Isg15+/+ and Isg15-/- mice. On 7.5 dpc, Isg15-/- mice showed reduced (P<0.001) uNK cell numbers and substantially reduced migration into the mesometrial pole of the decidua when compared to Isg15+/+ mice. By 12.5 dpc, there was a dramatic difference in the distribution of uNK cells in histological sections of Isg15-/- mice when compared with Isg15+/+. In Isg15-/- uNK cells were more concentrated in the decidua basalis in addition to an apparent impaired development of the metrial zone. The present study suggests that ISG15 may play a role in the recruitment of uNK cells in the uterus during early gestation. We suspect that faulty uNK migration and distribution contributes to embryonic mortality observed in ISG15-/- mice. (poster)