Quantification of Immune Cells in the Primate Corpus Luteum During the Menstrual Cycle; Increases During the Late Luteal Phase Indicate a Potential Role in Luteolysis.

Abstract

The potential role of the immune system in regulation of the mammalian ovary has primarily been investigated in domestic animal and rodent models; however the presence of immune cells in the human ovary has also been noted by immunohistochemistry. Recently, microarray-identified luteal mRNAs for immune regulatory components such as chemokines and cytokines, immune cell-associated genes, as well as gene signaling pathways associated with immune function were found to fluctuate during the lifespan of rhesus macaque corpora lutea (CL). To characterize immune cell populations in primate CL throughout the menstrual cycle, luteal tissue was collected from rhesus females at discrete intervals of the luteal phase, including early days 3-5 (n=3), mid days 6-8 (n=4), mid-late day 10 (n=4) and day 12 (n=3), as well as after onset of luteal regression (2 days n=2, and 3-4 days n=4, following a drop in serum progesterone levels to < 0.3 ng/ml). Luteal cells were enzymatically dispersed, counted and viability assessed. Wet weight (mg) of CL were significantly different across the luteal phase (P<0.03), but total numbers of viable cells did not differ between groups (P=0.8). Dispersed cells were then incubated with fluorescently-labeled antibodies specific for immune-cell surface proteins: CD11b (ITGAM; neutrophils including activated lymphocytes, granulocytes and monocytes), CD14 (monocytes/macrophages), CD16 (FCGR3; primarily natural killer cells as well as neutrophils and macrophages), and CD3 (CD3-epsilon/T cell receptor complex; T lymphocytes). Number of labeled cells for each CL (positive events in 10,000 cells counted) was assessed using a BD LSR II flow cytometry instrument for each antibody (FCS Express version 3, De Novo Software). A non-immune, isotype antibody matched for each cell surface specific marker was used to gate the positive cells within the CL. The number of positive events recorded for immune cells at each stage of the luteal phase was then analyzed by one-way ANOVA (SAS version 2.0). Both CD11b- and CD14-positive cells (neutrophils and macrophages) were constant until increasing significantly 3-4 days after onset of luteal regression (P<0.0001). Numbers of CD16-positive (natural killer cells) increased initially between early and mid luteal phase to plateau through the mid-late stages, until a further 3-fold increase by 3-4 days after onset of regression (P<0.005). Additionally, CD16-positive cells were the most abundant immune cell type in CL during the mid and mid-late luteal phases, and increased to levels comparable with CD11b-positive cells during luteal regression. Presence of CD3-positive lymphocytes tended to increase 3-4 days after regression (P=0.07), but were less abundant than other cell types. These data demonstrate a remarkable increase in cells of the innate immune system, (i.e. macrophages and neutrophils) in the primate CL associated with the latter stages of luteal regression. Natural killer cells may have a dual role in the CL to promote luteal structure and function during the mid-luteal phase, whereas macrophages, neutrophils and natural killer cells may participate in structural luetolysis at the end of the luteal lifespan. Supported by NIH grant R01HD020869 (RLS) and NCRR grant P51RR000163 (Support For National Primate Research Center).