**4.3. Ivmmune cells**

Regulation of the mammary gland morphogenesis also pertains to the involvement of immune cells and the utilization of immune-related signaling molecules [67]. The immune system may contribute to mammary development at each stage via cytokine secretion and recruitment of macrophages, eosinophils, neutrophils, mast cells, and lymphocytes (T and B cells) [48, 66]. The gland is intercalated with extensive vascular and lymphatic networks present throughout the fat pad. During pubertal mammary gland development, the lymphatic network develops in close association with the mammary epithelial tree and blood vasculature. The presence of immune cells within the surrounding stroma was shown to be important for ductal branching as these cells are recruited to the branching tips of the epithelium to mediate invasion into the fat pad [67].

### *4.3.1. Regulation of immune cells present within the mammary stroma*

Immune microenvironment of the mammary gland is also driven by the hypothalamicpituitary-gonadal axis. Hormones act directly on epithelial cells and may modulate immune impact on tissue remodeling. Estrogen, progesterone, and prolactin each regulate immune cell functions, which in turn support the morphogenic processes occurring in the pubertal and adult mammary gland [68]. The effects of these hormones on immune cells can be either direct or indirect. The direct effects are mediated when the immune cells express receptors for estrogen, progesterone, and prolactin, which are activated by their respective ligands. The indirect effects of these hormones on immune cells are mediated by paracrine signals derived from MECs and the surrounding stroma [66]. It has been shown that mice lacking the expression of estrogen receptor alpha (ERα) and amphiregulin (a member of EGF family) and showing deficient signaling driven by EGF receptor (ERBB1) fail to develop mature ductal trees and have inhibited recruitment of macrophages and eosinophils to the site of tissue remodeling [66]. In vitro experiments demonstrated that estrogen-stimulated macrophages significantly enhanced fibroblast proliferation and invasion by tumor necrosis factor (TNFα) and MMP9 secretion, thus modifying stromal tissue compartment for epithelial expansion [47].

The profile of immune cells within the microenvironment of the mammary gland varies depending on the changes in hormonal stimuli occurring during the estrus/menstrual cycle. In humans, during the luteal phase of the menstrual cycle, the dominant subpopulation of Th lymphocytes is the Th2 cells secreting IL-4 and IL-10. Increasing concentrations of estrogen increase the abundance of regulatory T cells (Treg) in blood and enhance their immunosuppressive functions [66]. It is speculated that since estrogen and progesterone regulate the number of Treg cells in blood, the abundance of these cells in the mammary gland may also be hormone-dependent and fluctuate over the menstrual cycle. In addition, progesterone during pregnancy and prolactin during lactation were shown to stimulate the recruitment of Th2 cells. These hormones induce MECs to produce Th2-like cytokines, such as IL4, IL5, IL10, and IL13 [69, 70].

#### *4.3.2. Role of eosinophils in mammary gland morphogenesis*

fibronectin, laminins, elastin, proteoglycans, and MMPs. Due to their properties, fibroblasts support the luminal epithelial growth and branching morphogenesis as well as participate in

Regulation of the mammary gland morphogenesis also pertains to the involvement of immune cells and the utilization of immune-related signaling molecules [67]. The immune system may contribute to mammary development at each stage via cytokine secretion and recruitment of macrophages, eosinophils, neutrophils, mast cells, and lymphocytes (T and B cells) [48, 66]. The gland is intercalated with extensive vascular and lymphatic networks present throughout the fat pad. During pubertal mammary gland development, the lymphatic network develops in close association with the mammary epithelial tree and blood vasculature. The presence of immune cells within the surrounding stroma was shown to be important for ductal branching as these cells are recruited to the branching tips of the epithelium to mediate invasion into the

Immune microenvironment of the mammary gland is also driven by the hypothalamicpituitary-gonadal axis. Hormones act directly on epithelial cells and may modulate immune impact on tissue remodeling. Estrogen, progesterone, and prolactin each regulate immune cell functions, which in turn support the morphogenic processes occurring in the pubertal and adult mammary gland [68]. The effects of these hormones on immune cells can be either direct or indirect. The direct effects are mediated when the immune cells express receptors for estrogen, progesterone, and prolactin, which are activated by their respective ligands. The indirect effects of these hormones on immune cells are mediated by paracrine signals derived from MECs and the surrounding stroma [66]. It has been shown that mice lacking the expression of estrogen receptor alpha (ERα) and amphiregulin (a member of EGF family) and showing deficient signaling driven by EGF receptor (ERBB1) fail to develop mature ductal trees and have inhibited recruitment of macrophages and eosinophils to the site of tissue remodeling [66]. In vitro experiments demonstrated that estrogen-stimulated macrophages significantly enhanced fibroblast proliferation and invasion by tumor necrosis factor (TNFα) and MMP9

secretion, thus modifying stromal tissue compartment for epithelial expansion [47].

The profile of immune cells within the microenvironment of the mammary gland varies depending on the changes in hormonal stimuli occurring during the estrus/menstrual cycle. In humans, during the luteal phase of the menstrual cycle, the dominant subpopulation of Th lymphocytes is the Th2 cells secreting IL-4 and IL-10. Increasing concentrations of estrogen increase the abundance of regulatory T cells (Treg) in blood and enhance their immunosuppressive functions [66]. It is speculated that since estrogen and progesterone regulate the number of Treg cells in blood, the abundance of these cells in the mammary gland may also be hormone-dependent and fluctuate over the menstrual cycle. In addition, progesterone during pregnancy and prolactin during lactation were shown to stimulate the recruitment of Th2 cells. These hormones induce MECs to produce Th2-like cytokines, such as IL4, IL5, IL10, and

the mammary gland tissue remodeling during involution (**Figure 2**).

102 Stromal Cells - Structure, Function, and Therapeutic Implications

*4.3.1. Regulation of immune cells present within the mammary stroma*

**4.3. Ivmmune cells**

fat pad [67].

IL13 [69, 70].

Eosinophils belong to immune cells found around the growing TEBs. These cells are attracted by eotaxin, another chemokine produced by mammary gland [43]. Eosinophil knockdown mice show altered elongation and branching during mammary gland development as well as insufficient milk productions at the time of lactation [48]. Similar abnormalities can be noted in knockout mice with deficiency of interleukin 5 (IL-5), a cytokine to which eosinophils are particularly responsive [71]. Mammary tissues from IL-5-deficient females had fewer TEBs, less well-branched mammary ducts, and lower overall density of the mammary gland structures. Furthermore, IL-5-deficient pups nursed by IL-5-deficient mothers were notably underweight, with a high percentage of pre-weaning mortality, in contrast to well-developed IL-5-deficient mice which were nursed by IL-5-sufficient foster mothers [71]. Interestingly, overabundance of eosinophils during puberty results in retarded morphogenesis of the mammary epithelium, suggesting the existence of mechanisms controlling the number of these cells that reside in the gland and are involved in MECs expansion during morphogenesis [66]. In addition to eosinophils, mast cells were also shown to be important for normal mammary gland development. Mice deficient in mast cells have defective mammary branching during puberty. It may be associated with the lack of vascular endothelial growth factor (VEGF) released by these cells that assist in mast cell degranulation [48]. Through activation of their serine proteases and degranulation, mast cells are involved in normal branching during puberty, and they accumulate and possibly activate plasma kallikrein, thus activating the plasminogen [5]. Furthermore, it was demonstrated that inhibition of this mast cell-associated protease during involution caused an accumulation of fibrillar collagen and delayed repopulation of adipocytes, thus preventing the gland from regaining the pre-pregnant state [14].

#### *4.3.3. Role of macrophages in mammary gland development and remodeling*

The role of macrophages at different stages of glandular morphogenesis as well as remodeling are better recognized. In the pubertal mammary gland, macrophages are recruited to the highly mitotic terminal end buds from which ducts elongate and branch to give rise to a mature ductal tree [48]. Macrophage colony stimulating factor-1 (CSF1) secreted by myoepithelial cells is a key cytokine that regulates the recruitment, proliferation, and survival of macrophages [48, 72]. Estrogen-regulated CSF1synthesis is essential for expanding of epithelial ducts and buds and alters structural alignment of collagen fibers around the expanding TEBs [70]. Macrophage abundance changes over the estrous cycle, peaking at metestrus and diestrus phases, and being the lowest at proestrus and estrus [66]. Studies on Csf1op/op mice, which are homozygous for a null mutation in *Csf1* gene, revealed that these animals exhibited multiple defects and had reduced macrophage numbers in most tissues including the mammary gland [72]. Depletion of mammary gland macrophages observed in Csf1op/op mice altered the mammary stem/progenitor cell activity, which was reflected in a substantially reduced outgrowth potential of the mammary epithelium. The mammary glands of Csf1op/op mice displayed lower number of TEBs as well as reduced ductal branching and elongation. During pregnancy, Csf1op/op glands developed precocious alveolar units but failed to switch to the lactational state resulting in impaired lactation [72]. These observations prove a continued requirement for normal macrophages during ductal morphogenesis and their stimulatory role on the putative basal progenitor cells. Macrophages also mediate the switch from pregnancy to lactation through regulation of tight junction permeability. In mice, activation of NF-κB by toll-like receptor 4 (TLR4) signaling pathway increases permeability of the milkblood barrier [66].

*4.3.4. Summary*

**4.4. Vascular endothelial cells**

mammary epithelium.

*4.4.1. Development of mammary blood vasculature*

The intricate interactions between immune and epithelial cells are an inherent part of the mammary gland physiology. Paracrine factors secreted by Th2 lymphocytes and macrophages (IL-4, IL-10, and TNFα) as well as direct crosstalk between MECs and macrophages, eosinophils, mast cells are involved in regulation of all stages of mammary gland morphogenesis, from early embryogenesis, puberty, through pregnancy, lactation and involution (**Figure 2**).

Stromal-Epithelial Interactions during Mammary Gland Development

http://dx.doi.org/10.5772/intechopen.80405

105

Mammary gland development, occurring during pre- and postnatal life of female mammals, serves to create a highly branched network of ducts and alveoli made of secretory epithelium that actively synthesizes and secretes milk at the time of lactation. To fulfill its function, the mammary gland also requires an expanded network of vascular endothelium. Currently, it is thought that the vasculature not only provides nutrients to the developing and functionally active mammary parenchyma, but also it is important for maintaining homoeostasis of the

Vasculature in the mammary gland undergoes repeated cycles of expansion and regression concomitantly with the cycles of growth, differentiation, and regression of the mammary epithelium [80]. The development of blood vessels occurs in parallel with mammogenesis. In the course of vascularization, first the process of de novo blood vessel formation takes place in the embryonic life, followed by angiogenesis which serves to form new vessels from pre-existing ones [80]. Angiogenesis is driven in main part by epithelial and stromal cells through secretion of the vascular endothelial growth factor (VEGF) and matrix metalloproteinases, especially MMP-9. Furthermore, studies have shown that development of the vessels in the mammary gland is driven by the same hormones that stimulate growth of the glandular

Before pregnancy, the mammary vasculature is composed of a thin layer of simple squamous endothelial cells forming a complex vascular network along with myoepithelial cells and connective tissue [82]. The structure of the glandular vasculature has been the best characterized in the mouse mammary gland. It is described as the basket-like capillary beds surrounding the alveoli clusters [83]. The capillary vessels run in parallel or encircle the mammary parenchyma and branch throughout the adipose tissue [82]. In humans, a high number of small capillaries are surrounding the ductal structures, whereas the acini of the lobular structures are interspersed by fewer, but significantly larger capillaries, which are sinusoidal in shape [80]. Such morphology provides a slower blood flow, thus a prolonged contact of the lobuloalveolar epithelium with circulating hormones and nutrients. During pregnancy, the growth of the mammary vessels intensifies along with expanded development of the parenchyma in order to increase the cell number and surface area to provide a maximal interface for nutrient transfer and milk secretion after parturition. Furthermore, increased surface area of the luminal endothelium is also accomplished by formation of microvilli and marginal folds

parenchyma, that is the metabolic and sex hormones and the growth factors [81].

Post-lactating involution, which is analogous to a wound healing response, involves complex stromal-epithelial interactions, activation of elements of both innate and adaptive immune system, as well as stimulation of inflammatory cytokines and proteinases expression. This process is mediated in part through Jak/Stat signaling pathway and is characterized by the apoptotic death of MECs and their removal and engulfing by phagocytic cells: macrophages and epithelial cells by process of efferocytosis [11]. Tissue resident and infiltrating macrophages have special role in that process. Specific depletion of these cells in the involuting mammary gland leads to a reduction in both lysosomal-mediated and apoptotic cell death [73]. Involution is associated with the polarization of macrophages away from proinflammatory (M1) phenotype to an alternatively activated state (M2) [74]. This phenotypic switch is STAT3-dependent and occurs within an infiltrating macrophage population from day 3 of involution [75]. Re-emergence of adipose tissue is an important feature of involution associated with infiltration of macrophages into the gland form [14]. In the mouse mammary gland, gene expression profiling during postlactational tissue regression showed an increase in genes linked to the immune system, which coincides with increasing levels of interleukins: IL-4 and IL-13 acting as macrophage chemoattractants [76]. Furthermore, ECM can fragment into matrikines and matricryptins that also serve as attractants for the peripheral immune cells [14]. Fragments of collagen I, collagen IV, laminins, and nidogen-1 have all been shown to promote chemotaxis of monocytes and neutrophils within the interstitial tissue. Once in the mammary gland, macrophages and neutrophils secrete proteases such as MMP9 and elastase that are involved in further ECM breakdown [73]. Thus, without the influx of macrophages or neutrophils, the remodeling of the mammary tissue during involution, that serves to return the gland to the non-secretory postpartum state, could be delayed or incomplete [14].

ECM fragments not only aid the immune cells infiltration into the mammary gland but also may act as ligands to receptors present on leukocytes residing in the mammary gland. Fragments of biglycan, heparan sulfate, and hyaluronan have been shown to act as ligands for toll-like receptor 4 (TLR4) [14]. Toll-like receptors (TLRs) are part of the pattern recognition receptor family expressed on the cell surface of innate immune cells and dendritic cells. Binding the ligand to its TLR activates the immune cell or induces secretion of cytokines by these cells, resulting in further activation of cells of the adaptive immune system. For example, binding of soluble biglycan TLR 2/4 on macrophages stimulates them to synthesize and release a proinflammatory cytokine interleukin-1β [77]. Other ECM components, such as heparan sulfate and hyaluronan, have been shown to bind to the TLR4 on dendritic cells, causing their maturation [78, 79]. In turn, mature dendritic cells are able to activate cells of the adaptive immune system, which migrate to the site of ECM remodeling [14]. Also the presence of B lymphocytes in involuting mammary gland may be connected with the chemoattractive properties of ECM fragments. In vitro studies revealed that interleukin-4 and fibronectin stimulated B cells motility, and both compounds are known to be upregulated during involution. In fact, the presence of B cells during early to mid involution has been confirmed, prior to the peak in macrophage recruitment [35].

#### *4.3.4. Summary*

pregnancy to lactation through regulation of tight junction permeability. In mice, activation of NF-κB by toll-like receptor 4 (TLR4) signaling pathway increases permeability of the milk-

Post-lactating involution, which is analogous to a wound healing response, involves complex stromal-epithelial interactions, activation of elements of both innate and adaptive immune system, as well as stimulation of inflammatory cytokines and proteinases expression. This process is mediated in part through Jak/Stat signaling pathway and is characterized by the apoptotic death of MECs and their removal and engulfing by phagocytic cells: macrophages and epithelial cells by process of efferocytosis [11]. Tissue resident and infiltrating macrophages have special role in that process. Specific depletion of these cells in the involuting mammary gland leads to a reduction in both lysosomal-mediated and apoptotic cell death [73]. Involution is associated with the polarization of macrophages away from proinflammatory (M1) phenotype to an alternatively activated state (M2) [74]. This phenotypic switch is STAT3-dependent and occurs within an infiltrating macrophage population from day 3 of involution [75]. Re-emergence of adipose tissue is an important feature of involution associated with infiltration of macrophages into the gland form [14]. In the mouse mammary gland, gene expression profiling during postlactational tissue regression showed an increase in genes linked to the immune system, which coincides with increasing levels of interleukins: IL-4 and IL-13 acting as macrophage chemoattractants [76]. Furthermore, ECM can fragment into matrikines and matricryptins that also serve as attractants for the peripheral immune cells [14]. Fragments of collagen I, collagen IV, laminins, and nidogen-1 have all been shown to promote chemotaxis of monocytes and neutrophils within the interstitial tissue. Once in the mammary gland, macrophages and neutrophils secrete proteases such as MMP9 and elastase that are involved in further ECM breakdown [73]. Thus, without the influx of macrophages or neutrophils, the remodeling of the mammary tissue during involution, that serves to return

the gland to the non-secretory postpartum state, could be delayed or incomplete [14].

confirmed, prior to the peak in macrophage recruitment [35].

ECM fragments not only aid the immune cells infiltration into the mammary gland but also may act as ligands to receptors present on leukocytes residing in the mammary gland. Fragments of biglycan, heparan sulfate, and hyaluronan have been shown to act as ligands for toll-like receptor 4 (TLR4) [14]. Toll-like receptors (TLRs) are part of the pattern recognition receptor family expressed on the cell surface of innate immune cells and dendritic cells. Binding the ligand to its TLR activates the immune cell or induces secretion of cytokines by these cells, resulting in further activation of cells of the adaptive immune system. For example, binding of soluble biglycan TLR 2/4 on macrophages stimulates them to synthesize and release a proinflammatory cytokine interleukin-1β [77]. Other ECM components, such as heparan sulfate and hyaluronan, have been shown to bind to the TLR4 on dendritic cells, causing their maturation [78, 79]. In turn, mature dendritic cells are able to activate cells of the adaptive immune system, which migrate to the site of ECM remodeling [14]. Also the presence of B lymphocytes in involuting mammary gland may be connected with the chemoattractive properties of ECM fragments. In vitro studies revealed that interleukin-4 and fibronectin stimulated B cells motility, and both compounds are known to be upregulated during involution. In fact, the presence of B cells during early to mid involution has been

blood barrier [66].

104 Stromal Cells - Structure, Function, and Therapeutic Implications

The intricate interactions between immune and epithelial cells are an inherent part of the mammary gland physiology. Paracrine factors secreted by Th2 lymphocytes and macrophages (IL-4, IL-10, and TNFα) as well as direct crosstalk between MECs and macrophages, eosinophils, mast cells are involved in regulation of all stages of mammary gland morphogenesis, from early embryogenesis, puberty, through pregnancy, lactation and involution (**Figure 2**).
