Maternal milk is a complex biological fluid principally viewed as a food providing energy and essential nutrients for growth and development of newborns. However, milk also contains a multitude of bioactive enzymes, hormones, growth factors and immunological agents with diverse biochemical specificities (Ellis and Picciano 1992, Goldman et al. 1996). Many of these agents were originally viewed as insignificant byproducts of milk secretion, because they are present in milk at less than picogram quantities. These components are typically abundant in the early milk when significant functional immaturity of neonatal organ systems exists. Such temporal relationships suggest that bioactive milk constituents may be important in neonatal development. Available evidence to support the function of milk-borne cytokines and hormones as developmental regulators of the immune system in neotates is presented.

NEONATAL IMMUNODEVELOPMENT

Lymphocytes from neonatal humans and rats are not readily activated (Nehlesen-Cannarella and Chang 1992); their ability to reject tissue grafts is poor, and clonal expansion (i.e., proliferation) of circulating lymphocytes, thymocytes and splenocytes following challenge with polyclonal mitogens in vitro is lower in comparison with adults (Grove et al. 1991, Middleton and Bullock 1984). The limited capacity of lymphocytes to become activated is a consistently observed feature of the immune response of neonates that is due in part to active suppression of lymphocyte proliferation and in part to the reduced capacity of immune cells of neonates to synthesize regulatory cytokines. Natural killer cells are present in small numbers and, similarly, their functional responses in neonates are very low.

Intraepithelial lymphocytes and Peyer's patches reside within the small intestine (Guy-Grand et al. 1993) and differentiate extensively in the postnatal period as neonates are exposed to environmental antigens. Cells maturing in the Peyer's patches are capable of migrating from the intestine to secondary immune organs (Ottaway et al. 1987), whereas the intraepithelial lymphocytes do not migrate but respond to antigenic substances present locally in the intestine.

MATERNAL MILK CONSTITUENTS WITH IMMUNOMODULATORY ACTIVITY AND THEIR POTENTIAL ROLES IN NEONATES

If milk constituents with immunomodulatory activity are important for neonatal system maturation, one would expect neonates deprived of these factors to exhibit impaired immune system function. At least two lines of evidence suggest that the neonatal immune system may be influenced by immunomodulatory agents in maternal milk. First, in vitro and ex vivo studies showed that several aspects of the acquired or antigen-driven immune response differ between human neonates fed maternal milk and those fed substitutes devoid of immunomodulatory activity (Stephens 1991). Second, results of epidemiological studies suggest that human milk feeding confers protection against acute and chronic diseases such as respiratory synctial virus, otitis media, diarrhea and diseases related at least in part to disorders of the immune system, i.e., autoimmune disorders (Crohn's disease and diabetes mellitus) and lymphoma (Goldman and Goldblum 1995). Moreover, the protection afforded by maternal milk feeding is evident not only during infancy but also well into adulthood (Ellis and Picciano 1992).

PROLACTIN IS AN IMMUNOMODULATORY AGENT

BIOLOGICALLY ACTIVE PROLACTIN IS PRESENT IN MILK

In addition to PRL variants present in the glycosylated/phosphorylated fraction of milk, high-molecular-weight forms of PRL are detected in milk of rabbits (Postel-Vinay et al. 1991), humans (Mercado and Baumann 1994) and rats (Ellis et al. 1996). Very-high-molecular-weight or "big-big" and "big" forms of PRL have been described in other biological fluids and are known to originate from the association of 23-24-kDa PRL or its variants with PRL-R, PRL-binding proteins or immunoglobulin or through PRL dimerization. The secretion of PRL into milk complexed with binding proteins/receptors may be an important mechanism for protecting milk PRL from proteolytic degradation and for preserving PRL biological activity in the intestinal tract of neonates.

DEMONSTRATION OF MILK PROLACTIN ACTIVITY IN NEONATES

EVIDENCE THAT MILK-PROLACTIN INFLUENCES NEONATAL IMMUNE CELL FUNCTION

In another study, the proliferation response and CD marker expression of thymocytes and splenocytes of 5-d-old mice ingesting PRL-poor milk since birth were also examined (Gala and Shevach 1993). Comparison of these results to those for neonatal rats is confounded because immune endpoints were examined at an earlier age (d 5) in neonatal mice than in rats (d 10). Nonetheless, neonatal mice fed PRL-poor milk showed significantly reduced splenocyte numbers of d 5, although differences in proliferation and differentiation marker expression were not yet evident. Administration of anti-PRL serum directly to neonatal mice and rats has also been used to reduce levels of serum PRL while maintaining pituitary PRL synthesis. Thymocytes and splenocytes of suckling mice treated with PRL-antiserum on d 1-3 of life were characterized by significant reductions in the percentages of CD4+ T-helper lymphocytes and the percentage of B cells expressing immunoglobulin G (Russell et al. 1988). Thus, data from neonatal rats and mice suggest that ingestion of PRL-poor milk may affect differentiation of immune cells in the thymus, migration of thymocytes to secondary immune organs as well as proliferation of thymocytes and splenocytes when challenged with polyclonal mitogens in vitro. The manisfestation of these alterations are unknown; they may not be evident during or immediately following the period of reduced milk PRL ingestion and may be evident only as the neonate matures.

Prolactin exerts its biological effects through PRL-R signal transduction. Both thymocytes and splenocytes express long and short forms of the PRL-R at birth (Gunes and Mastro 1996). Prolactin receptor expression in the thymus does not change significantly during the period from birth to early adulthood, but in the spleen it increases during the first 2 wk of life (Fig. 2 A, B). The expression of PRL-R in both the spleen and thymus of neonates is down-regulated by ingestion of milk (Fig. 2 C, D). Levels of PRL-R in the spleen decrease 7 h after birth in neonates ingesting milk compared with littermates not ingesting milk (Fig. 2 C, D). The down-regulation of PRL-R expression in the spleen of neonates by milk feeding may be one mechanism by which milk PRL regulates immune cell function and influences lymphocyte trafficking. Most recently, we observed that intraepithelial lymphocytes express high levels of PRL-R in the neonatal period, and we are investigating whether ingestion of milk PRL influences PRL-R expression in this cell population.

SUMMARY AND FUTURE DIRECTIONS FOR RESEARCH

Although the role of immunomodulatory agents in the development of the immune system of neonates is presently awaiting detailed exploration, there is sufficient evidence to warrant such exploration. Prolactin is just one of many agents with immunomodulatory activity in maternal milk, and it is likely that other milk cytokines exert their pleiotropic actions on neonatal target tissues. The immediate goals for defining the immunomodulatory roles of milk cytokines and hormones, however, include isolating the factors from milk, defining target cells affected by these agents in neonates, and establishing in vitro model systems for their study. The form or activity of milk-borne cytokines and hormones in blood of neonates may differ from their form or activity present in maternal milk, and therefore the role of the gastrointestinal tract as a possible modifier of milk cytokines and hormones must be evaluated. Until these goals are met, the potential effect of milk immunomodulatory agents in human neonates, particularly those born prematurely, cannot be assessed.

ACKNOWLEDGMENTS