Requirement of macrophages and eosinophils and their cytokines/chemokines for mammary gland development

Abstract

Epithelial/mesenchymal cell interactions are necessary for proper ductal morphogenesis throughout all stages of mammary gland development. Besides the well-established stromal components, such as adipocytes and fibroblasts, the mammary stroma is also infiltrated with migrating blood cells, mostly macrophages and eosinophils. The focus of this review is on the role of macrophages and their growth factor colony-stimulating factor 1 (CSF-1) in promoting branching morphogenesis during postnatal mammary gland development through to lactation. The more restricted role of eosinophils and their chemoattractant eotaxin during pubertal ductal morphogenesis is also discussed. A possible interaction between macrophages and eosinophils in ductal morphogenesis is considered, along with the roles of other chemokines. This role of macrophages in normal development also appears to be subverted by tumors of the mammary gland to promote the escape of the tumor cells from the local environment and enhance their rate of metastasis. These data emphasize the dual role of macrophages in the promotion of epithelial growth in normal and cancer states.

Figure 1

Macrophage and eosinophil distribution during mammary gland development and differentiation. (a-e) Longitudinal paraffin section of a terminal end bud (TEB) was stained twice, first with hematoxylin/eosin (H/E) (d) and then, after destaining, by immunostaining with anti-F4/80 antibody and counterstaining with hematoxylin (a-c). The F4/80⁺ cells were detected with a peroxidase-coupled detection system (brown coloration). (b,c,d) High-magnification pictures of (a): (b) bottom frame; (c,d) top frame. The pictures of doubly stained cells show the cross-reactivity of anti-F4/80 antibody for macrophages (b, filled arrowheads), mainly seen in the neck of the TEB (bottom frame, a) and for eosinophils (c,d, empty arrowheads), mainly seen around the head of the TEB (top frame, a). F4/80⁺ eosinophils were distinguished from F4/80⁺macrophages by their characteristic eosin-pink cytoplasmic granules (d, empty arrowheads) and their segmented nucleus, sometimes in a ring shape with a round cytoplasmic shape (b,c,d, empty arrowheads). In contrast, F4/80⁺ macrophages have a large, oval nucleus with a spread cytoplasm (b,c, filled arrowheads) containing no eosin-pink cytoplasmic granules. (e) The presence of macrophages inside the TEB, where they engulf apoptotic epithelial cells. (f-h) F4/80 immunostaining of mammary sections at day 16 of pregnancy (f,g) and day 2 postpartum (h). Note the abundance of macrophages tightly associated with the lobulo-alveoli during pregnancy and lactation. Original magnification: a, f, h, 400×; b, c, d, e, g 1000×. Panels a-e modified from [7].

Figure 2

Leukocytes are necessary for mammary ductal outgrowth. (a,b) +/Csf1^op mice were irradiated (700 rads, gamma-irradiation) at 19 days of age. A representative mammary gland whole-mount preparation obtained 10 days after irradiation is illustrated (a), in comparison with the nonirradiated control (b). (c,d) +/Csf1^op mice underwent gamma-irradiation (700 rads) at 19 days of age and 2 hours later were (c) or were not (d) given a transplant of a bone marrow cell suspension from +/Csf1^op mice. Representative whole-mount preparations obtained 28 days after irradiation/transplantation (c) and from an irradiated control mouse that did not undergo bone marrow transplantation (d) are shown. Note the restoration of ductal outgrowth after bone marrow transplantation. All the photomicrographs shown are of whole mounts from the entire fourth abdominal mammary gland and were taken at the same magnification. Modified from [7].

Figure 3

Macrophage distribution in mammary glands of +/Csf1^op control and Csf1^op/Csf1^op mice during pregnancy and lactation. F4/80 immunohistochemistry of mammary section of day 14 (P14; a-d) and day 18 (P18; e,f) of pregnancy and day 4 postpartum of lactation (PP4; g,h) from control (a,c,e,g) and Csf1^op/Csf1^op (b,d,f,h) mice. Note the abundance of F4/80⁺ macrophages tightly associated with alveoli in control mice at all stages shown, while macrophages are almost absent in Csf1^op/Csf1^op mice at all stages of mammary development. The few macrophages (filled arrowheads, d) seen in Csf1^op/Csf1^op mice are not adjacent to the alveoli, and most of the F4/80 cells seen are eosinophils (empty arrowheads, d). Original magnification: a,b,e,f,g,h: × 400; c,d: × 1000.

Figure 4

Model for macrophage potentiation of normal mammary gland development and for tumor progression. CSF-1/chemokines produced by the TEB or tumor regulate infiltration and activity of macrophages. These cells release growth factor remodeling signals and angiogenic factors that promote vascularization and/or invasion of mammary epithelial or tumor cells through the matrix. Ang, angiopoietin; EGF, epidermal growth factor; MMP, matrix metalloproteinase; TNF, tumor necrosis factor; uPA, urokinase plaminogen activator; VEGF, vascular endothelial growth factor.

Figure 5

C10 expression in mammary gland. (a, b) C10 Immunohistochemistry of a longitudinal section of a TEB. C10⁺ cells are detected with a peroxidase-coupled detection system (brown coloration). (b) High magnification of the frame seen in (a). Note the positive eosinophils in the stroma surrounding epithelial cells of the TEB, characterized by their segmented nucleus, sometimes in a ring shape, with a round cytoplasmic shape. Original magnification: a, 400×; b 1000×.