Immunology of Human Milk

Antibodies

Human milk contains immunoglobulin (Ig)M, IgG, IgD, and IgA. The concentration of IgM is much lower in human milk than in serum. IgM molecules in blood and milk are pentamers. However, unlike serum IgM, most human milk IgM is bound to a secretory component, and the antibody specificities are often similar to those of human milk secretory IgA (SIgA). Concentrations of IgG in human milk are lower than those for IgM and are much lower than serum IgG levels. All IgG subclasses are found in human milk, but the proportion of IgG4 is higher in milk. Very little IgD is present in human milk. IgE is undetectable.

In contrast, SIgA comprises more than 95% of human milk immunoglobulins. Most SIgA consists of two identical IgA monomers united by a 15-kDa polypeptide called the joining chain and complexed to secretory component, a 75-kDa glycopeptide fragment of the polymeric Ig receptor on the mammary gland epithelium. ^, SIgA is assembled when dimeric IgA produced by plasma cells in the stroma of the mammary gland binds to the first domain of polymeric Ig receptors on the basolateral surface of the mammary gland epithelium. The complex is internalized, the cytoplasmic part of the receptor is cleaved off, and the remaining protein (SIgA) is transported into milk.

SIgA antibodies in human milk are principally directed against foreign enteric and respiratory microbial antigens ( Table 122.2 ). Antibody-producing cells in the mammary gland originate from those mucosal sites, where their precursors are released by antigen stimulation. During lactation, antigen-stimulated B cells from Peyer patches (aggregated lymphoid nodules) in the lower small intestines switch from producing IgM to dimeric IgA and then migrate to the mammary gland. ^, A similar B-cell pathway links bronchial lymphoid tissues to the mammary gland.

The switch from IgM ⁺ to dimeric IgA ⁺ in B cells in Peyer patches requires cytokines produced by local mononuclear leukocytes. These isotype-switched B cells migrate sequentially into local lymphatics, mesenteric lymph nodes, the cisterna chyli, the thoracic duct, and blood. CCL28 and its receptor CCR10 are crucial to this process. CCL28 is up-regulated in the murine mammary gland epithelium during lactation. Most dimeric IgA ⁺ B cells in the enteromammary gland pathway display CCR10, the receptor for that chemokine. CCL28 expressed by mammary gland epithelium is a chemoattractant for dimeric IgA ⁺ CCR10 ⁺ B cells.

After entering the mammary gland, dimeric IgA ⁺ B cells differentiate into dimeric IgA-producing plasma cells in the lamina propria. IgA dimers produced by those plasma cells principally contain λ-light chains, whereas κ-light chains predominate in serum immunoglobulins. IgA dimers bind to polymeric Ig receptors on the basolateral external membranes of mammary gland epithelial cells. ^{, , ,} The resultant complex is transported to the apical side of an epithelial cell where the original intracytoplasmic portion of the receptor is cleaved away. The resultant molecule, SIgA, is secreted into milk. Thus enteromammary and bronchomammary pathways protect the infant against pathogens in the environment of the dyad (see Table 122.2 ). This is important because SIgA antibodies and the antigen-binding repertoire of immunoglobulins are not optimally produced during early infancy. Further, antiidiotypic SIgA antibodies in human milk elicit an immune response as though they were the original foreign antigens.

Some of the natural SIgA antibodies in human milk, which arise without antigenic stimuli, are directed against CCR5, the co-receptor for R5-tropic strains of human immunodeficiency virus (HIV)-1. Via CCR5, macrophages and immature dendritic cells become infected with HIV-1. Antibodies from HIV-1-infected women bind to the second extracellular loop of CCR5 and thus reduce HIV-1 infection of macrophages and dendritic cells. In addition, SIgA antibodies to DNA enzymatically cleave DNA. ^, Therefore, the DNA is recognized not only as an autoantigen but also as a substrate by the antibody-enzyme that hydrolyzes free nucleic acids in the recipient’s intestinal and respiratory tracts.

The quantity of SIgA in human milk gradually declines as lactation proceeds, but considerable SIgA is transmitted to the infant throughout breast-feeding. Concentrations of SIgA in human milk are highest in colostrum and gradually plateau later in lactation. The approximate mean daily intake of SIgA in healthy, term, breast-fed infants is 600 mg/day at 1 month and 500 mg/day by 4 months.

The pattern of immunoglobulins in human milk differs from that in other mammals except for closely related primates. For example, IgG is the dominant immunoglobulin in bovine colostrum, and much of it is absorbed by the calf, whose IgG production is developmentally delayed. Without colostrum, calves are IgG-deficient and susceptible to intestinal infections.

Human SIgA is resistant to intestinal proteases including pancreatic trypsin. Bacterial proteases attack the hinge region of IgA1, but IgA2 is resistant to those proteases and is disproportionally increased in human milk. Furthermore, SIgA antibodies against bacterial IgA proteases are in human milk. Consequently, the amount of SIgA excreted in stools of low-birth-weight infants fed human milk is approximately 30 times that in infants fed a cow’s milk formula. In addition, urinary excretion of SIgA rises as a result of human milk feedings. ^, It is unlikely that the antibodies are from human milk, because there is no mechanism for their transport from the gastrointestinal tract to the blood. The mechanism responsible for SIgA antibodies in the infant’s urinary tract remains unclear.

Lactoferrin

Lactoferrin is a glycoprotein with two globular lobes, each of which displays a binding site for ferric iron. In 90% of lactoferrin in human milk, iron-binding sites are free to compete with siderophilic bacteria and fungal enterochelin for ferric iron. Iron chelation disrupts proliferation of those pathogens. Lactoferrin also kills by damaging outer membranes of many gram-positive and gram-negative bacteria and filamentous fungi by a peptide comprised of 18 amino acid residues from the N-terminal region formed by pepsin digestion (lactoferricin). ^, Furthermore, lactoferrin inhibits certain viruses in a chelation-independent manner. With free secretory component, lactoferrin interferes with the adhesion of Escherichia coli to epithelial cells.

The mean concentration of lactoferrin in human colostrum is between 5 and 6 mg/mL. The concentration falls to approximately 1 mg/mL at 2 to 3 months of lactation. The mean intake of milk lactoferrin in healthy breast-fed term infants is approximately 1200 mg/day at 1 month and 700 mg/day by 4 months. Because human lactoferrin resists proteolysis and the concentration of lactoferrin is much greater in human than bovine milk, the excretion of lactoferrin in the stools is higher in infants fed human milk than in those fed cow’s milk. ^, The quantity of lactoferrin excreted in stools of low-birth-weight infants fed human milk is approximately 185 times that excreted by infants fed cow’s milk. However, that estimate may be too high because fragments of lactoferrin, which may be biologically active, are present in stools of human milk-fed infants. There is also a significant increment in urinary excretion of intact and fragmented lactoferrin as a result of human milk feedings. ^, The increase is due to absorbed human milk lactoferrin and its fragments.

Lysozyme

Lysozyme, a 15-kDa single chain protein, lyses susceptible bacteria by hydrolyzing β-1,4 linkages between N -acetylmuramic acid and 2-acetylamino-2-deoxy- d -glucose residues in cell walls. High concentrations of lysozyme are in human milk throughout lactation. Longitudinal changes in lysozyme during lactation are unlike most other agents in human milk. The mean concentration of lysozyme is approximately 70 μg/mL in colostrum, 20 μg/mL at 1 month, and 250 μg/mL by 6 months of lactation. The reason why the concentration rises by 6 months is not understood. The high content of lysozyme in human milk and its resistance to proteolysis lead to an eight-fold increase in lysozyme excreted in the stools of low-birth-weight infants fed human milk as compared to infants fed cow’s milk. However, urinary excretion of lysozyme is not increased in infants fed human milk.

α-Lactalbumin

α-Lactalbumin is expressed only in the lactating mammary gland. A folding variant of the protein kills Streptococcus pneumoniae . Furthermore, multimeric α-lactalbumin kills certain tumor cells in vitro by inducing apoptosis. ^,

CCL28

CCL28 kills Candida albicans and many gram-positive and gram-negative bacteria. The killing is mediated by its 28 amino acid carboxyl terminus.

Macrophage Migration Inhibitory Factor

Macrophage migration inhibitory factor, a constituent of human milk, is a proinflammatory cytokine (see later) that also up-regulates TLR-4 and aids in killing Mycobacterium tuberculosis in macrophages.

Fibronectin

Fibronectin, which is found in human milk, facilitates uptake of bacteria and certain other particulates by mononuclear phagocytes. Its in vivo effects in human milk are unknown.

Complement

All components of the classic and alternative pathways of complement are in human milk, but their concentrations are much lower than those in serum. In vitro experiments suggest that C3 in human milk augments phagocytosis of microbial pathogens in the gastrointestinal and respiratory tracts.