411. Macrophage abundance and phenotype in the mammary gland fluctuates throughout the estrous cycle

Abstract

Macrophages are versatile cells with roles in the generation and execution of immune responses, tissue breakdown and remodelling and production of regulatory cytokines. These cells are implicated in development of reproductive tract tissues that mature after birth. Descriptive and knockout studies indicates that macrophages play multiple roles in epithelial cell proliferation, phagocytosis and tissue remodelling during pubertal mammary gland development, pregnancy, lactation, and involution. However, the role of macrophages in remodelling over the course of the ovarian cycle has not been previously investigated. Oestrus cycles were monitored in C57/Bl6 mice by daily vaginal smears, and mammary glands were analysed for the abundance and location of macrophages in all four stages of the cycle (n = 6–8 per group). Absolute number of macrophages was quantified by FACs analysis of digested mammary gland tissue stained with macrophage-specific antibody F4/80. The location of macrophages was investigated in fixed, paraffin embedded tissue by immunohistochemistry using F4/80 antibody. H&E staining revealed that the most differentiated epithelial ducts and alveoli structures appeared in diestrus and proestrus. Whilst there was no difference in the absolute number of macrophages at any stage, the location of these macrophages varied. Macrophages accumulated around the more highly differentiated epithelial ducts, leading to significantly more macrophages around the ducts during proestrus compared with metestrus (P < 0.05). These macrophages were in close contact with the epithelium, and displayed long projections and widespread cytoplasm. In contrast, macrophages on the edge of the gland within the collagen-rich connective tissue were characteristically round in shape. These results indicate that macrophages are present in the adult virgin mammary gland, and fluctuate in location and phenotype over the course of the oestrous cycle. Further analysis of the phenotypes of macrophages present in these different locations is required for a more comprehensive understanding of the role of macrophages in mammary tissue homeostasis.