Host-Resistance Factors and Immunologic Significance of Human Milk

Variation in Breast Milk Cell Content

Early studies demonstrated macrophages, lymphocytes, neutrophils, and epithelial cells in breast milk totaling approximately 4000/mm. ¹⁰² , ¹⁰³ Cell fragments and epithelial cells were examined by electron microscope in fresh samples from 30 women by Brooker. ¹⁰⁴ He found that the membrane-bound cytoplasmic fragments in the sedimentation pellet outnumbered intact cells. The fragments were mostly from secretory cells that contained numerous cisternae of the rough endoplasmic reticulum, lipid droplets, and Golgi vesicles containing casein micelles. Secretory epithelial cells were found in all samples. Ductal epithelial cells were about 1% of the population of cells for the first week or so and then disappeared. All samples contained squamous epithelial cells, originating from lactiferous ducts and the skin of the nipple.

More recent data reinforce the concept that the cell content of human milk varies by mother, and by lactation stage (colostrum, early lactation, late lactation). The estimated range of cells is between 10,000 and 13,000,000 cells/mL ( Fig. 5.3 ). ⁸⁷ , ⁸⁸ , ⁹² , ⁹⁹ Hassiotou et al. reported variation in numbers of cells and cell composition with milk fat content, after removal of milk, and during occurrence of infection in the mother or infant. ⁸⁸ , ¹⁰⁵ With this heterogenous pattern of cells in breast milk, leukocytes remain the dominant cell type in colostrum and the milk of early lactation and epithelial cells dominate in mature milk. ⁸⁸ , ⁹² , ⁹⁹ , ¹⁰³ , ¹⁰⁵

Leukocytes

Living leukocytes are normally present in human milk. ⁸⁵ The overall concentration of these leukocytes is of the same order of magnitude as that seen in peripheral blood, although the predominant cell in milk is the macrophage rather than the neutrophil. Macrophages compose about 40% to 50% of the leukocytes, and 2000 to 3000/mm ³ are present, followed by polymorphonuclear neutrophils, also 40% to 50%. ¹⁰⁵ , ¹⁰⁶ Lymphocytes make up about 5% to 10% of the cells (200 to 300/mm ³ ), which is a much lower concentration than in human blood. ¹⁰⁷ The number of leukocytes found in human milk increases with mastitis and with evidence of infection in the mother or infant and then decrease to baseline at resolution. ¹⁰⁶ , ¹⁰⁸ Both large and small lymphocytes are present. By indirect immunofluorescence with anti–T-cell antibodies to identify thymus-derived lymphocytes, it has been shown that 50% of human colostral lymphocytes are T cells and up to 80% of the lymphocytes in human milk are T cells. ¹⁰⁹ Immunofluorescence procedures to detect surface immunoglobulins characteristic of B lymphocytes identified 4% to 6% as B lymphocytes. ¹⁰⁹ , ¹¹⁰

The number of leukocytes and the degree of mitogenic stimulation of lymphocytes sharply decline during the first 2 or 3 months of lactation to essentially undetectable levels, according to Goldman et al. ¹¹¹ ( Fig. 5.4 ). More recent studies using new cell counting and identification techniques demonstrate that mature milk actually contains less than 2% leukocytes in a healthy mother–infant dyad. ⁸⁸ , ¹⁰⁵ Hassiotou et al. calculated the daily intake of leukocytes by an infant by breast milk to be hundreds to thousands based on the normal daily infant intake of milk, the reported range of total cell count in breast milk, and this 2% fraction of cells. ⁸⁷ , ⁸⁸

Macrophages

Macrophages are large-complex phagocytes that contain lysosomes, mitochondria, pinosomes, ribosomes, and a Golgi apparatus. The monocytic phagocytes are lipid laden and were previously called the colostral bodies of Donne . They have the same functional and morphologic features as phagocytes from other human tissue sources. These features include ameboid movement, phagocytosis of microorganisms (fungi and bacteria), killing of bacteria, and production of complement components C3 and C4, lysosome, and lactoferrin. Other milk macrophage activities include the following: ¹¹²

• Phagocytosis of latex, adherence to glass

• Secretion of lysozyme, complement components

• C3b-mediated erythrocyte adherence

• IgG-mediated erythrocyte adherence and phagocytosis

• Bacterial killing

• Inhibition of lymphocyte mitogenic response

• Release of intracellular IgA in tissue culture

• Giant cell formation

• Interaction with lymphocytes

Data suggest these macrophages also amplify T-cell reactivity by direct cellular cooperation or by antigen processing. The colostral macrophage has been suggested as a potential vehicle for the storage and transport of immunoglobulin. A significant increase in IgA and IgG synthesis by colostral lymphocytes, when incubated with supernatants of cultured macrophages, has been reported. ¹⁰³

The macrophage may also participate in the biosynthesis and excretion of lactoperoxidase and cellular growth factors that enhance growth of intestinal epithelium and maturation of intestinal brush-border enzymes.

The mobility of macrophages is inhibited by the lymphokine migration inhibitor factor, which is produced by antigen-stimulated sensitized lymphocytes. The activities of macrophages have been demonstrated in both fresh colostrum and colostral cell culture. Certain functions are altered compared with their counterpart in human peripheral blood.

Polymorphonuclear Leukocytes

The highest concentration of cells occurs in the first few days of lactation and reaches more than a million per milliliter of milk. This correlates with the period of highest basement membrane permeability in the infant’s gut. Some authors propose that this decline of cells and specifically leukocytes is related to the improved intestinal barrier and diminished need for leukocytes.

Colostrum (1 to 4 days postpartum) contains 10 ⁵ to 5×10 ⁶ leukocytes/mL, and 40% to 60% are polymorphonuclear cells (PMNs). Mature milk (i.e., after 4 to 5 days) has fewer cells ( Fig. 5.5 ), approximately 10 ⁵ /mL with 20% to 30% PMNs. After 6 weeks, fewer PMNs are present. The functions of the PMNs normally include microbial killing, phagocytosis, chemotactic responsiveness, stimulated hexose monophosphate shunt activity, stimulated nitroblue tetrazolium dye reduction, and stimulated oxygen consumption. ¹¹³ When milk PMNs are compared with those in the serum, their activity is often less than that of serum PMN cells. Whether milk PMNs actually perform a role in the protection of the infant has been studied by many investigators using many techniques. Briefly, animal studies have shown that (1) the mammary gland is susceptible to infection in early lactation, (2) a dramatic increase in PMNs occurs with mammary inflammation, and (3) in the presence of peripheral neutropenia during chronic mastitis, severe infection of the gland occurs. This implies, according to Buescher and Pickering, that the primary function of milk PMNs is to defend the mammary tissue, per se, and not to impart immunocompetence to the newborn. ¹¹³ This may explain the presence of large numbers of PMNs that are relatively hypofunctional early and then disappear over time. Evidence shows that neutrophils found in human milk demonstrate signs of activation, including increased expression of CD11b (an adherence glycoprotein), decreased expression of L-selectin, spontaneous production of granulocyte-macrophage colony-stimulating factor (GM-CSF), and the ability to transform into CD1 ⁺ dendritic cells (DCs). ¹¹⁴ Human milk macrophages have the morphology and motility of activated cells. The movement of these cells in a three-dimensional system is greater than that of monocytes, their counterparts in peripheral blood. Such activated neutrophils may play a role in phagocytosis at the level of the mucosa of the GI tract, supplementing infants’ poor ability to recruit phagocytes to that site. ¹¹⁵

Lymphocytes

Both T and B lymphocytes are present in human milk and colostrum and are part of the immunologic system in human milk. T cells are 80% of the lymphocytes in breast milk. Human milk lymphocytes respond to mitogens by proliferation, with increased macrophage-lymphocyte interaction and the release of soluble mediators, including migration inhibitor factor. Cells destined to become lymphopoietic cells are derived from two separate influences, the thymus (T) and the bursa (B) or bursal equivalent tissues. The population of the B cells makes up the smaller part of the total. They synthesize IgA antibody. The term B cell is derived from its origination in a different anatomic site from the thymus; in birds, it has been identified as the bursa of Fabricius. The B cells can be identified by the presence of surface immunoglobulin markers. The B cells in human milk include cells with IgA, IgM, and IgG surface immunoglobulins. B cells transform into plasma cells and remain sessile in the tissues of the mammary gland.

T Cell System

More rapid mitotic activity occurs in the thymus gland than in any other lymphatic organ, yet 70% of the cells die within the cell substance. The thymus is the location for much of the T cell differentiation and selection and plays a major role in the development of infants’ immune systems. Thymosin has been identified as a hormone produced by thymic epithelial cells to expand the peripheral lymphocyte population. After emergence from the thymus gland, T cells acquire new surface antigen markers. The T cells circulate through the lymphatic and vascular systems as long-lived lymphocytes, which are called the recirculating pool. They then populate restricted regions of lymph nodes, forming thymic-dependent areas. ¹¹⁰ It is interesting to note that exclusively breastfed infants have a significantly larger thymus than formula-fed infants at 4 and 10 months. ¹¹⁶ The significance of the lymphocytes in human milk in affording immunologic benefits to breastfed infants continues to be investigated. It is suggested that lymphocytes can sensitize, induce immunologic tolerance, or incite graft-versus-host reactions. According to Head and Beer, lymphocytes may be incorporated into sucklings’ tissues, achieving short-term adoptive immunization of the neonate. ¹¹⁷ Cabinian et al. described in a mouse model breast milk T lymphocytes and cytotoxic T cells (CTLs) localizing in Peyer’s patches of the mouse pup's intestine. ¹¹⁸

Studies of the activities of lymphocytes have been carried out by a number of investigators who collected samples of milk from lactating women at various times postpartum, examined the number of cell types present, and then studied the activities of these cells in vitro. ⁸⁵ , ⁸⁶ , ¹¹⁹ Ogra and Ogra ¹²⁰ collected samples from 200 women and measured the cell content from 1 through 180 days (see Fig. 5.5 ). They then compared the response of T lymphocytes in colostrum and milk with that of the T cells in the peripheral blood. T cell subpopulations also have been shown by surface epitopes to be similar to those in the peripheral blood.

The greatest number of cells appeared on the first day, with the counts ranging from 10,000 to 100,000/mm ³ for total cells. By the fifth day, the count had dropped to 20% of the first day’s count. In addition, the number of erythrocyte rosette-forming cells was determined by using sheep erythrocyte-rosetting technique. The erythrocyte rosette formation lymphocytes constituted a mean 100/mm ³ on the first day and one-tenth of that by the fifth day.

At 180 days, total cells were 100,000/mm ³ , lymphocytes were 10,000/mm ³ , and erythrocyte rosette formation lymphocytes were 2000/mm ³ . The investigators compared the values with those in the peripheral blood of each mother; the levels remained essentially constant. ⁸⁵ In a similar study, Bhaskaram and Reddy ¹²⁰ sampled milk over time from 74 women and found comparable cell concentrations. They examined the bactericidal activity of the milk leukocytes and found it to be comparable with that of the circulating leukocytes in the blood, irrespective of the stage of lactation or state of nutrition of the mother. ¹²⁰

Ogra and Ogra also studied the lymphocyte proliferation responses of colostrum and milk to antigens. ¹²¹ Their data show response to stimulation from the viral antigens of rubella, cytomegalovirus (CMV), and mumps. Analysis of cell-mediated immunity to microbial antigens shows milk lymphocytes are limited in their potential for recognizing or responding to certain infectious agents compared with cells from the peripheral circulation. This is thought to be an intercellular action and not caused by lack of external factors. In contrast, the T cells and B cells have been shown to have unique reactivities not seen in peripheral blood.

Colostral lymphocytes are derived from mature rather than immature T-cell subsets. The distribution of T-cell subsets in colostrum includes both CD4 ⁺ and CD8 ⁺ cells. ¹²² The distribution of CD4 cells in colostrum and human milk is lower than in the serum, and fewer CD4 cells exist than CD8 cells. The percentage of CD4 cells is higher than in the serum of either postpartum donors or normal control subjects. No correlation exists with length of gestation and number of cells (normal blood usually contains twice as many CD4 ⁺ as CD8 ⁺ lymphocytes). ¹²³

Parmely et al. partially purified and propagated milk lymphocytes in vitro to study their immunologic function. ¹²⁴ Milk lymphocytes responded in a unique manner to stimuli known to activate T lymphocytes from the serum. The authors found milk lymphocytes to be hyporesponsive to nonspecific mitogens and histocompatibility antigens on allogenic cells in their laboratory. They found them unresponsive to Candida albicans . Significant proliferation of lymphocytes occurred in response to the K1 capsular antigen of Escherichia coli . ¹²⁵ Lymphocytes from blood failed to respond to the same antigen. This supports the concept of local mammary tissue immunity at the T-lymphocyte level.

More recent experiments in rodents have provided evidence that T lymphocytes that are reactive to transplantation alloantigens can adoptively immunize a suckling newborn. Foster nursing experiments performed in rodents have shown that newborn rats exposed to allogenic milk manifested alterations in their reactivity to skin allografts of the foster mother’s strain. In animals, mothers may give their suckling newborn immunoreactive lymphocytes. The influence of maternal milk cells on the development of neonatal immunocompetence has been demonstrated in several different immunologic contexts. Congenitally, athymic nude mice nursed by their phenotypically normal mothers or normal foster mothers had increased survival. The mothers contributed their T-cell helper activity to the suckling newborn.

Colostral lymphocytes proliferate in response to various mitogens, alloantigens, and conventional antigens. Colostral cells survive in the neonatal stomach and in the gut of experimental animals, some remaining viable in the upper GI tract for a week. No evidence, however, indicates that transepithelial migration takes place when neonatal mice are foster-nursed by newly delivered animals whose colostral cells were tagged with H-thymidine. ¹¹³

Cells in human milk have been studied using the same markers employed with cells in the peripheral blood; 80% of the lymphocytes are T cells that are equally distributed between CD4 ⁺ and CD8 ⁺ subpopulations, and their T-cell receptors are principally of the α/β type. CD4 ⁺ cells are common leukocyte cells of the helper and suppressor-inducer subsets, and CD8 ⁺ cells are leukocytes of cytotoxic and noncytotoxic subsets. T cells in human milk are presumed activated because they display increased phenotypic markers of activation, including human leukocyte antigen (HLA)-DR and CD25 (IL-2 receptor). The majority of T cells in human milk are CD45RO ⁺ , consistent with effector and memory T cells. ¹⁰⁹ , ¹²⁶ These cells are effective producers of IFN-γ, which is consistent with their phenotypic features. Here again, human milk may supplement the infant with a functioning immune cell to compensate for an identified deficiency in the infant, a paucity of memory T cells.

B Cell System

Juto studied the effect of human milk on B-cell function. ¹²³ Cell-free, defatted, filtered colostrum, as well as mature breast milk, showed an enhancing effect on B-cell proliferation and generation of antibody secretion. This was not seen with formula. Juto ¹²³ suggested that this could represent an important immunologic mechanism. Goldblum et al. ¹²⁷ demonstrated a B-cell response in human colostrum to E. coli given to the mother orally, which was not accompanied by a systemic response in the mother. This suggests that the breast and breast milk reflect sites of local, humoral, or cell-mediated immunity, which were initially induced at a distant site such as the gut and transferred by reactive lymphoid cells migrating to the breast. Head and Beer provided a scheme to describe this mechanism (see Fig. 5.1 ). ¹¹⁷ The diagram depicts the progeny of specifically sensitized lymphocytes that originated in GALT, specifically Peyer’s patches, as they migrate to the mammary gland. As they infiltrate the mammary gland and its secretion, they supply the breast with immune cells capable of selected immune responses. Ogra and Ogra suggest that the cells may selectively accumulate in the breast during pregnancy. ⁸⁵ , ¹²¹ The responses of milk cells and their antibodies are not representative of an individual’s total immunity. ¹²⁴ Most of these immunocompetent cells, initially stimulated in GALT, recirculate to the external mucosal surface and populate the lamina propria as antibody-producing plasma cells. A substantial number of these antigen-sensitized cells selectively home-in to the stroma of the mammary glands and initiate local IgA antibody synthesis against the antigens initially encountered in the respiratory or intestinal mucosa. ¹²¹ More recent work on human milk–derived B cells demonstrates that breast milk contains activated memory B cells, different from those in the blood. These cells express mucosal adhesion molecules ( ${\alpha }_4{\beta }_7^{-/+}$ , ${\alpha }_4{\beta }_1^{+}$ , CD44 ⁺ , CD62L ⁻ ), suggesting an origin in the mammary gland, but similar to GALT-associated B cells. ¹²⁸ The mucosae-associated epithelial cytokine CCL28 may contribute to migration of and retention of these cells in the mammary gland. ¹²⁹ This information supports the concept of the mammary gland as an effector site of the mucosal immune system.

The accumulated epidemiologic research supports the concept that colostrum and milk provide human infants with immunologic benefits. Both T and B lymphocytes found in breast milk are reactive against organisms invading the intestinal tract. However, the proof of specific viral or bacterial protection, secondary to the action of immunologically active B cells, has not been demonstrated.

Mammary Gland Cells

The mammary gland of female mammals is a remarkable organ, only reaching full maturation after birth. It undergoes monthly changes because of the hormonal fluctuations associated with the menstrual or estrous cycle and the more remarkable changes in structure and function through pregnancy, lactation, and involution. This dramatic morphogenesis along with the gland’s importance in the production of breast milk and nurturing of mammalian infants has led to renewed study of its anatomy, histology, development and functioning. ⁸⁷ , ⁹⁰ , ⁹¹ , ⁸² , ⁹³ , ¹⁰¹ Separately the cellular origin and evolution of breast cancer is driving other avenues of research. ⁹⁴ , ⁹⁵ , ⁹⁶ , ¹³⁰ , ¹³¹

The mammary gland as a functional organ consists of a number of different cell types: epithelial, adipose, immune, lymphatic, fibroblasts, vascular cells, and presumably progenitor cells/stem cells. The cells commonly identified in the greatest numbers in breast milk are epithelial cells, bacterial cells, hematogeneous immune cells, and, in fewer numbers, progenitor stem cells ⁹⁴ , ⁹⁵ , ⁹⁶ , ¹³⁰ (see Fig. 5.2 .) Epithelial cells (ductal and alveolar, luminal-epithelial, and myoepithelial are essential to the lactating breast and can comprise over 90% of the cells in the breast milk of a healthy mother and infant. ⁹¹ , ¹⁰⁵ Lactocytes are thought to predominate in mature human milk, although the exact percentage range of lactocytes varies from 10% to 28% to 11% to 99% in different studies. ⁸⁷ By comparison, in cow’s milk the macrophage is the predominant cell type. ⁸⁷ The majority of the epithelial cells are viable in freshly acquired breast milk. ⁹⁸ , ¹⁰⁵ That raises the questions of why epithelial cells are present in breast milk and what function they serve. Do the cells enter breast milk as the result of apoptosis or the mechanical forces of synthesis, secretion, and ejection or expression (manual or pump) or an active process driven by gene expression to create “mobile” cells entering the breast milk with specific functions? ¹³² Epithelial cells are noted to form clusters in breast milk and are cultivable. ¹⁰⁴ It is not just the luminal ductal or alveolar epithelial cells that are found in breast milk but also myoepithelial cells from the basal epithelial layers of ducts and alveoli. ⁹¹ , ⁹⁷ How and why do basal epithelial cells enter breast milk? Notably various researchers have proposed that breast milk contains cells from the full spectrum of mammary epithelial cells differentiation. This includes multipotent stem cells, luminal and ductal stem cells, luminal and ductal progenitors, and the more differentiated epithelial cells. ⁸⁷ , ⁹² , ⁹³ Ongoing flow cytometry studies with cell marker identification, molecular analysis, genomics, and even single cell RNA sequencing studies are revealing new information about the mammary epithelial cell developmental stages and its cellular function and potential. ⁹² , ⁹³

Stem Cells

Interest in mammary stem cells (MaSCs) has blossomed since Cregan et al. ⁹⁷ reported the presence of MaSCs in human breast milk. Their research was based on the demonstration of the cytokeratin 5 MaSC marker on cells isolated from human breast milk. Additional analysis showed cells from human milk with both the multipotent stem cell marker nestin and the cytokeratin 5 marker. There are several areas of interest relative to these MaSCs in humans: the potential readily availability of multipotent mesenchymal stem cells (MSCs) for autologous stem cell therapies; the identified cell markers and signaling pathways active in these cells, which could lead to more targeted breast cancer therapies; the role of stem cells in the dynamic states of the breast, especially lactation; the potential correlation between MaSCs and transplantation tolerance; and the state of microchimerism of MaSCs in the infant and the potential effects on the infant. ⁸⁷ , ⁹⁸ , ⁹⁹ , ¹³³ , ¹³⁴ , ¹³⁵ , ¹³⁶ , ¹³⁷ , ¹³⁸

The mammary gland is an attractive target in the search for stem cells, in that it is a dynamic, metabolically active tissue. It has the capacity to proliferate and hypertrophy through adolescent development, pregnancy, lactation, and the subsequent involution phase of the breast. Human embryonic stem cells (hESCs) have a tremendous differentiation potential, in that they can develop into every cell type in the body, different from adult stem cells, which constitute a small portion of organ cells and can mature into organ-specific cell types. Adult stem cells presumably can also produce new stem cells to maintain the population of these cells within the organ. They are said to remain quiescent within “stem cell niches” within an organ. ¹³⁸ Hassiotou et al. described human breast milk stem cells (hBSCs) with evidence of pluripotency markers on these cells similar to those on hESCs. ⁹¹ , ⁹⁸ The hBSCs were different from the hESCs in that they did not form tumors in the teratoma assay. ⁹⁸ Additional analysis of hBSCs by the same group demonstrated that they are capable of differentiating into the mammary cell lineage (myoepithelial cells and lactocytes) and cells of all three germ layers (ectoderm, mesoderm, endoderm), including hepatocytes, adipocytes, chondrocytes, osteoblasts, cardiomyocytes, neurons. ⁸⁸ , ⁹⁸ Other investigators demonstrated what appear to be MSCs also in human breast milk. ⁹⁹ There is a question of whether these represent true MSCs or evidence of epithelial to mesenchymal transition occurring in the breast. The full complement of stem cells or progenitor cells in human breast milk remain to be fully identified. Inman et al. ⁹² and Witkowska-Zimny et al. ⁸⁹ reviewed the various mammary gland cells identified to date in human breast milk, including stem cells, progenitor cells, mammary epithelial cells, and even hematopoietic stem cells in small numbers (see Fig. 5.2 ). Subsequent research has identified signaling pathways related to stem cell propagation, including Wnt/beta-catenin, Notch, Hedgehog (Hh) transforming growth factor-β (TGF- β), phosphatase, tensin homologue, and Bmi. ¹³⁸ Stem cells have many of the features of tumor cells, including self-renewal and the ability to replicate “indefinitely.” ^138,139 The question is what might distinguish normal progenitor cells from tumorigenic progenitor cells. Other investigators searching for such tumorigenic mammary gland stem cells identified MaSCs with the surface markers Lin ⁻ CD29hiCD24 ⁺ , which were capable of generating a functional mammary gland in the mouse. ¹³⁷

The potential roles of hBSCs remain to be determined. The most likely of these roles is in the mother directly contributing to the changes from stage to stage of breast development in pregnancy to lactation. In the infant, the roles could include setting up a microchimerism state leading to local tissue homeostasis or regeneration and tolerance to various maternal antigens. The exact mechanism of acquired tolerance to noninherited maternal antigens (NIMAs) is unknown. It has been suggested that exposure of the fetus during pregnancy and exposure during breastfeeding to NIMA may be the explanation for transplantation tolerance in breastfed persons. ¹⁴⁰ , ¹⁴¹ , ¹⁴² Breast milk contains a variety of major histocompatibility complex (MHC) antigens from the mother. Molitor et al. ¹⁴¹ demonstrated high levels of NIMA HLA proteins in both the cord blood and breast milk, emphasizing the potential role of human breast milk in exposing the infant to NIMAs. Dutta and Burlingham ¹⁴³ propose that stem cell microchimerism in infants is related to tolerance, specifically to NIMAs.

There remains much more to be understood about the existence of human breast stem cells in human milk and their possible role in health in the infant and later in life.

Mammary Cells and Breast Cancer

Breast cancer is a very heterogenous disease. The World Health Organization has defined 18 different subtypes based on histologic and clinical features. There also have been 5 molecular subtypes described: luminal A, luminal B, HER2 positive [HER2+], basal-like, and normal-like. They are distinguished by the expression of estrogen receptors (ER + or –), progesterone receptors (PR + or –), HER2 expression, and gene expression signature similar to or dissimilar from “normal mammary gland tissue.” ^95,130 The different subtypes of breast cancer demonstrate different survival rates and response to treatment. It is this cellular and molecular heterogeneity combined with the high mortality, recurrence rates, drug resistance, and metastases, which is pushing research to characterize tumorigenesis of breast cancer. There are two main theories of breast cancer: the stem cell hierarchy model and the clonal evolution model. ¹³⁷ The stem cell hierarchy model says there are “malignant” stem cells, which lead to cancer (cancer stem cells [CSCs]), resulting in a small group of tumorigenic cells and a predominance of nontumorigenic cells that differentiate from the stem cells, leading to the heterogeneity and a hierarchy of differentiated cells in the tumor. The clonal evolution model states that individual cells develop genetic and epigenetic changes over time that lead to cellular characteristics, giving them a selective advantage over other cell clones (i.e., phenotypic and functional differences facilitating tumor growth and survival). This can create a homogenous or heterogenous phenotype of cancer cells. In the CSC model, therapy could be targeted at primarily the tumorigenic cells, and in the clonal evolution model, therapy would need to target most of the cells.

The overlap between breast cancer pathogenesis and mammary gland functional biology is understanding the roles of stem cells in the breast and breast milk (hBSCs and progenitor stem cells), and the regulatory pathways, mammary gland microenvironments, hormonal effects, and noncoding RNAs that influence normal proliferation, differentiation, and apoptosis related to the normal maturation and functioning of the mammary gland. ⁹⁶ Some of the regulatory/signaling pathways being studied include Wnt/Beta-catenin, Notch, Hedgehog, signal transducer, activator of transcription-5a and -5b (STAT5), and the p53 pathway. The mammary gland microenvironment as it influences normal stem cell function and differentiation involves signaling from extracellular matrix molecules, stromal-derived growth factors, proteolytic enzymes, cytokines, and steroid hormones. This same environment when altered could lead to tumorigenesis. Noncoding RNAs (transcribed from the genome, but not encoding proteins, ncRNAs) can affect gene expression by targeting mRNA and influencing cell proliferation and differentiation and stem cell maintenance within microenvironments. ⁹⁶ Cataloging and comparing breast cancer phenotypes, outcomes, and cellular characteristics with the epigenomics, genomics, metabolomics, and cell lineage studies of breast cancer can lead to that enhanced understanding of breast cancer development and treatment. Ideally, this will also lead to understanding how a longer duration of breastfeeding confers protection against breast cancer.

Immunoglobulins

All classes of immunoglobulins are found in human milk. The study of immunoglobulins has been enhanced through the techniques of electrophoresis, chromatographics, and radioimmunoassay. More than 30 components have been identified; of these, 18 are associated with proteins in the maternal serum, and the others are found exclusively in milk. The concentrations are highest in the colostrum of all species, and the concentrations change as lactation proceeds. ¹⁴⁶ , ¹⁴⁷ IgA, principally sIgA, is highest in colostrum. Although postpartum levels fall throughout the next 4 weeks ( Fig. 5.6 ), substantial levels are maintained throughout the first year, during gradual weaning between 6 and 9 months, and even during partial breastfeeding (when the infant receives solid foods) in the second year of life ( Table 5.3 ). Specific sIgA antibodies to E. coli persist through lactation and may even increase (see Fig. 5.6 ).

The main immunoglobulin in human serum is IgG, and IgA content is only one-fifth the level of IgG. In milk, however, the reverse is true. IgA is the most important immunoglobulin in milk, not only in concentration but also in biologic activity. sIgA is likely synthesized in the mammary alveolar cells or by lymphocytes that have migrated from Peyer’s patches in the GI tract or from lymphoid tissue in the respiratory tract via the lymphatics to the breast. ¹⁴⁸ Cytokines cause isotype switching of local IgM ⁺ B cells to become IgA ⁺ B lymphocytes. ⁵⁴ , ¹⁴⁹ , ¹⁵⁰ These isotype switched cells travel to the breast, where they are transformed into plasma cells producing secretory, dimeric IgA. It is through this “enteromammary” pathway that the mother provides increased amounts of sIgA to the infant against the microorganisms present in the mother’s and infant’s environment. ⁷⁸ , ⁸² Brandtzaeg ¹⁵¹ has proposed a model for the transport of IgA (polymeric) and IgM (pentameric), produced by plasma cells, across the secretory epithelium. The model involves the formation of sIgA and IgM, through binding, with the secretory component attached to the epithelial membrane. This occurs in the membrane of mammary epithelial cells during lactation. ^151,152

Quantitative determinations of immunoglobulins in human milk were made from milk collected at birth to as long as 27 months postpartum by Peitersen et al. ¹⁵³ and by Goldman et al. ¹¹¹ The IgA content was high immediately after birth, dropping in 2 to 3 weeks, and then remaining constant. Similar observations were made on IgG levels and IgM levels. Ogra and Ogra compared serum and milk levels at various times postpartum. Samples obtained separately from the left and right breasts showed similar values. ^85,121 The levels remained constant during a given feeding and throughout a 24-hour period. In all quantitative determinations, IgA is the predominant immunoglobulin in breast milk, constituting 90% of all the immunoglobulins in colostrum and milk.

Ogra and Ogra studied the serum of postpartum lactating mothers and nonpregnant matched control subjects. They noted that the individual and mean concentrations of all immunoglobulin classes were lower in the postpartum subjects. The levels were statistically significant for IgG; they were 50 to 70 mg higher in the nonpregnant women. ^85,121,154 Immunoglobulin levels, particularly IgA and IgM, are very high in colostrum and drop precipitously in the first 4 to 6 days, but IgG does not show this decline. The volume of mammary secretion, however, increases dramatically in this same period; thus the absolute amounts of immunoglobulins remain more nearly constant than it would first appear. Local production and concentration of IgA, and probably IgM, may take place in the mammary gland at delivery.

IgE and IgD have been measured in colostrum and milk. Using radioimmunoassay techniques, colostrum was found to contain concentrations of 0.5 to 0.6 IU/mL IgE in 41% of samples and less in the remainder. ¹⁵⁵ IgD was found in all samples in concentrations of 2 to 2000 mg/dL. Plasma levels were poorly correlated. The findings suggest possible local mammary production rather than positive transfer. The question of whether IgE or IgD antibodies in breast milk have similar specificities for antigens as the IgA antibodies in milk remains unanswered. ¹⁴⁶ Keller et al. examined the question of local mammary IgD production, and its possible participation in a mucosal immune system, by comparing colostrum and plasma levels of total IgD with specific IgD antibodies. ¹¹⁹ From their work comparing colostrum-to-plasma ratios for IgG, IgD, and albumin and measuring IgD against specific antigens, the authors reported evidence for IgD participation in the response of the mucosal immune system, with increases in total IgD and IgD against specific antigens found in colostrum.

Butte et al. addressed the question of total quantities of immunologic components secreted into human milk per day and available to an infant. ¹⁵⁶ They did so by measuring the amounts of sIgA, sIgA antibodies to E. coli , protein, lactoferrin, and lysozyme ingested per day and per kilogram per day in the first 4 months of life ( Figs. 5.7 through 5.11 ). Lactoferrin, sIgA, and sIgA antibodies gradually declined in amount ingested per day and per kilogram per day. Lysozyme, in contrast, rose during the same period in total amount available and amount per kilogram per day. The authors suggest that production and secretion of these immunologic factors by the mammary gland may be linked to the catabolism of the components in an infant’s mucosal tissues. ¹⁵⁶ When the concentrations of sIgA, IgG, IgM, α ₁ -antitrypsin, lactoferrin, lysozyme, and globulins C3 and C4 were compared in relationship to parity and age of the mother, no consistent trend was observed. When maturity of the pregnancy was considered, however, mean concentrations of all these proteins were higher, except for IgA, when the delivery was premature. Because several proteins in human milk have physiologic functions in infants, Davidson and Lönnerdal ¹⁵⁷ examined the survival of human milk proteins through the GI tract. ¹⁵⁷ Crossed immunoelectrophoresis showed that three human milk proteins transversed the entire intestine and were present in the feces: lactoferrin, sIgA, and α ₁ -antitrypsin.

Miranda et al. reported on the effect of maternal nutritional status on immunologic substances in human colostrum and milk. ¹⁵⁸ Maternal malnutrition was characterized as lower weight-to-height ratio, creatine-to-height index, total serum proteins, and IgG and IgA. In malnourished mothers, the colostrum contained one-third the normal concentration of IgG, less than half the normal level of albumin, and lower IgA and complement C4. Lysozyme, complement C3, and IgM levels were normal. Levels improved with development of mature milk and improvement in maternal nutrition. According to one report in 2003, moderate exercise during lactation does not affect the levels of IgA, lactoferrin, or lysozyme in breast milk. ¹⁵⁹ Immunologic components contained in human milk during the second year of lactation become a significant point as more infants are nursed longer. For a longitudinal study of lactation into the second year by Goldman et al., women were included who had fully breastfed their infants for 6 months to a year and were continuing to partially breastfeed. ¹⁶⁰ Samples were collected by fully emptying the breast by electric pump. Table 5.3 summarizes the concentrations of the measured factors in breast milk from 12 to 24 months of lactation. No leukocytes were detected. Concentrations of total IgA and sIgA, lactoferrin, and lysozyme were similar to those 7 to 12 months postpartum and during gradual weaning. sIgA antibodies to E. coli were produced in the second year, demonstrating significant immunologic benefit to the infant with continued breastfeeding. ¹⁶⁰ IgA, IgM, and IgG were measured in nursing women from the beginning of lactation and simultaneously in the feces of their children by Jatsyk et al. at the Academy of Medicine in Moscow. ¹⁶¹ They reported IgA to be very high in the milk and rapidly increasing in the feces. IgG and IgM levels, however, were low in both milk and feces. In normal full-term bottle-fed infants, IgA appeared in the feces at 3 to 4 weeks of age, but at much lower levels than in breastfed infants. Koutras and Vigorita ¹⁶² reported that in the first 8 weeks of life increased amounts of sIgA were found in the stools of breastfed infants compared with formula-fed infants. The authors ascribed this phenomenon to the presence of sIgA in human milk and a stimulation of the local GI production of immunoglobulin.

Savilahti et al. measured serum levels of IgG, IgA, and IgM in 198 infants at 2, 4, 6, 9, and 12 months of age. ¹⁶³ By 9 months, the exclusively breastfed infants had IgG and IgM levels significantly lower than those who had been weaned early (before 3.5 months) to formula. Six infants were still exclusively breastfed at 12 months, and their IgA levels had also lowered to levels found at 2 months with bottle feeders. Infection rates were similar. Two months after the children were weaned to formula, the IgG and IgM levels were comparable. Iron and zinc levels were the same in all children.