Research Paper Mediators of Inflammation, 9, 133–140 (2000)

In breast milk and paired serum from 70 lactating women and 40 of their term, infection-free neonates, on the 2nd and 5th day postpartum slCAM-1, sVCAM-1, sE- and sL-selectin were measured by ELISA and compared with those in 26 healthy adults (controls). Seven infant formulas and fresh milk from five cows were also analyzed. Human colostrum values of slCAM-1, sVCAM-1 (similar to those in maternal and control serum), sE-selectin and sL-selectin (-10 and -100 times lower than in maternal and control serum) were significantly higher than those in milk, while they varied widely. None of the adhesion molecules was detected in fresh cow's milk or infant formulas. Exclusively breast-fed infants showed significantly higher values of slCAM-1 and sL-selectin on the 2nd day of life than those supplemented also with formula. Only slCAM-1 values correlated positively between colostrum and time-matched maternal serum. These findings show in human milk important amounts of slCAM-1 and sVCAM-1 but minimal amounts of sE- and sL-selectin, which could affect the immune system of the neonate.


Introduction
It is widely accepted that much of the protective effect of human milk is due not only to a direct-acting antimicrobial system that consists of humoral and cellular compounds but also to some immunomodulatory and immunoregulatory agents. 1 Among them proinflammatory and antiinflammatory cytokines have been extensively studied in the last 10 years. 2 -7 As regards soluble adhesion molecules, however, only a few sporadic references on soluble intercellular adhesion molecule-1 (slCAM-1), soluble vascular cell adhesion molecule-1 (sVCAM-1) and soluble E-selectin (sE-selectin) could be found in the literature. 8 -10 So far, to the best of our knowledge, soluble L-selectin (sL-selectin) has not been studied in human milk. For this reason, the aim of this study was to evaluate the presence of slCAM-1, sVCAM-1, sE-and sL-selectin in human milk during the first days of lactation and to examine whether these adhesion molecules are an intact part of the human breast milk immune system, their putative origin, as well as their possible immunomodulatory effects on the development and maturation of the neonatal immune system.

Subjects and sample collection
This study was approved by the Institutional Review Board for Human Research at our Teaching Hospital.
Maternal informed consent was also obtained. All participants were non-allergic, healthy lactating women (n = 70) of mean age (range) 27.8  years and had delivered vaginally (n = 53) or by elective cesarean section (n = 17) single, mature, healthy, appropriate for gestational age infants (29 females/41 males), after full term (38.8 weeks, 37.0-42.0), uncomplicated pregnancies. Thirty-seven of them were nulliparous and the remaining 33 multiparous.
A peripheral venous blood sample and a milk sample were collected on the 2nd and the 5th day postpartum. The milk samples were obtained by manual expression, just after venipuncture for the blood sample, one hour after last feeding in the morning. Furthermore, venous blood (~1 ml) from 40 of their 70 neonates was also obtained simultaneously with blood and milk sample collection from their mothers. Twelve neonates out of the 40 on the 2nd day of life were exclusively breast fed, whereas the remaining 28 were breast fed and supplemented with bovine milk-based infant formula. On the 5th day of life only 10 neonates were exclusively breast fed.
All neonates, lactating mothers and controls were infection free as judged by clinical examination and C-reactive protein (CRP) values (<3.5 mg/L), measured in the same blood samples as adhesion molecules.

Preparation of serum and milk samples
Peripheral venous blood samples were obtained in polypropylene pyrogen-free tubes and were kept at 5-8°C for clotting. Immediately after clotting, serum was separated by centrifugation in two steps in a refrigerated centrifuge at 2500 rpm for 10 min, aliquoted in five specimens and kept at -30° until assayed.
Milk specimens were collected also in pyrogen-free tubes and they were refrigerated immediately at 2-5°C for two hours. Then they were centrifuged in two consecutive steps, 2000 rpm for 10 min and 2500 rpm for 15 min at 6°C, to remove the cells and lipid layer. The defatted and cell-free aqueous layer of colostrum and milk samples was aspirated through the needle of a sterilized syringe and was passed through a special filter with pore diameter 0.45 mm (Minisart, Sartorius, Goetingen, Germany), to remove any last membrane-associated molecules and lipid droplets. Then cell-free aqueous fractions were aliquoted in small pyrogen-free tubes, and kept at -30°C until assayed.
A serum, colostrum and transitional milk sample from every lactating woman as well as age-matched serum samples of her neonate were analysed in the same run.

Statistical analysis
For the statistical analysis of the results, the statistical program Statgraphics, version 1.0 (Manugistics Inc, Rockville, MD, USA) was used. The frequency distribution of slCAM-1, sVCAM-1, sE-and sL-selectin values in neonatal, maternal and control serum was normal, while that in milk samples was not (Kolmogorov-Smyrnov test). For this reason, results in sera are given as mean values (±SD), whereas in milk as median values (upper and lower quartile). Data in serum were analysed using parametric methods (Student's t-test or paired t-test, as appropriate, if natural pairing occured in experimental series). Comparisons with and between milk samples were done using non-parametric methods: Wilcoxon test for unpaired measurements or paired Wilcoxon test (Wilkoxon signedranks test), as appropriate. Correlations of every adhesion molecule values between colostrum and milk samples or between them and the time-matched maternal or neonatal serum samples were examined by the non-parametric Spearman rank correlation coefficient (r S ), while between serum samples by the parametric Pearson correlation coefficient (r P ). The effect of the perinatal factors, mode of delivery, maternal age and parity or neonatal gender and birthweight on every adhesion molecule in breast milk, maternal or neonatal serum was examined by multiple regression analysis.

Mediators of Inflammation
In maternal serum: The study of correlations of the four adhesion molecules in breast milk, maternal and neonatal agematched serum samples revealed that:

Discussion
In this study we measured the serum adhesion molecules sICAM-1, VCAM-1, sE-and sL-selectin in the defatted and acellular aqueous phase of human colostrum and transitional milk during the first days of lactation. Our findings reveal that (a) colostrum and transitional milk contain important amounts of sICAM-1 and sVCAM-1 but minimal amounts of SEand SL-selectin, (b) significantly higher concentrations of all four adhesion molecules are found in human colostrum on the 2nd day compared with those in milk samples on the 5th day portpartum and (c) all four adhesion molecules studied present large variations in their concentrations in both colostrum and milk samples. Similar results have been reported for sICAM-1, sVCAM-1 and sE-selectin in a small number of human colostrum (n = 10) and milk (n = 13) samples and for sICAM-1 only in a very recent report 13 and an abstract. 12 To the best of our knowledge sL-selectin in human milk that we detected in 90% of the human colostrum samples and 80% of the transitional milk samples is reported here for the first time.
The significantly lower human milk content in adhesion molecules than that in colostrum led some investigators to suggest that sICAM-1, sVCAM-1 and sE-selectin were unlikely to contribute significantly in human milk's anti-inflammatory effects. 13 We believe, however, that the important amounts of sICAM-1 and sVCAM-1 in colostrum from the 2nd day postpartum suggest that at least these compounds could affect the recipient infant, particularly in the first days of life. On the other hand, the significantly higher concentrations of all four adhesion molecules in human colostrum (2nd day) compared with those in milk (5th day postpartum), as well as the higher concentrations of sICAM-1 in colostrum than in controls and lactating mothers, seem to be in analogy with the protein content of human colostrum and later breast milk, like albumin, protein hormones, and growth factors. 14,15 During the first 24 hr postpartum the colostrum production is ~120 mL, whereas on the 5th day the total volume/day of transitional milk reaches 250-300 mL. 16 Consequently, the total amount of sICAM-1/day in colostrum should be ~5000 to 187 500 ng/day, considering that concentrations range from 40 to 1500 ng/ml, while on the 5th day postpartum the total amount in milk should bẽ 6500-175 000 ng/day, considering that concentrations range from 26 to 700 ng/ml. Regarding even sLselectin, the respective content of colostrum per day should be 850-1560 ng and that in milk 500-1875 ng/ day, considering that the respective concentrations were 6.8-12 ng/ml and 2-7.5 ng/ml, respectively. Hence, we consider that physiologically important amounts per day of all four adhesion molecules are intaken by newborn infants through breast-feeding and could potentially act as immunomediators 8,10 or angiogenetic 17 and maturational agents. They might protect the newborn against infections of respiratory and intestinal tract and suppress certain diseases such as atopy, coelial disease 18 and inflammatory diseases of the neonatal gut during the first days of life, when there is increased risk of overshooting immune reactions, leading maybe to chronic inflammation, as proposed for certain cytokines and their soluble receptors. 6,9,13 They could also promote the growth, differentiation and maturation of very important neonatal tissues like gut and lung, and induce developmental and maturational processes of the immune system in neonates, similarly to milk hormones, cytokines and growth factors. 3,6,7,19 sICAM-1 and sVCAM-1, found in human milk in considerable amounts, do not differ significantly in maternal serum from those in controls. In contrast, sEand sL-selectin, which show very low concentrations in human milk, present in maternal serum significantly lower concentrations than those in controls. Matthiesen e t a l. have suggested that a state of systemic suppression of the maternal immune system seems to be present during pregnancy and the early postnatal period. 20 Values of soluble cell adhesion molecules in human milk were compared and correlated with those in time-matched maternal serum in order to get an indication of the origin of these biomolecules in human milk. However, for sICAM-1 values only, a significant positive trend between colostrum and time-matched maternal serum was noticed, similarly to a previous study. 13 Thus, it appears that only sICAM-1 could be derived at least partially directly from maternal circulation. Possible sources of the studied adhesion molecules in human milk could be cellular components such as leukocytes, macrophages, some T cells, vascular or epithelial cells found abundantly epecially in early milk. This latter observation is probably an explanation of the higher values in colostrum than in transitional milk. They could also derive from the cellular activity of the mammary gland itself, where components such as IgA, complex proteins and carbohydrates are synthesized. Further studies are needed to explore this hypothesis.
Our finding of a significant positive correlation between sICAM-1 and sVCAM-1 in colostrum is not in agreement with the results of a previous report; 11 this may be due to the very small number of the colostrum samples they analysed.
The low human milk content of selectins might be attributed to the transient expression of E-selectin, which peaks and begins to decline before the appearance of CD35 + cells. 10 In contrast with our results showing that all four adhesion molecules were found in human milk in quantities similar to (slCAM-1 and sVCAM-1), or less than (sE-selectin, sL-selectin) those in control serum, none of the four adhesion molecules could be detected in the reconstituted seven different bovine milk-based formulas, or in the fresh milk samples of five cows, treated with exactly the same protocol as human milk samples. It is possible that cow's milk contains adhesion molecules with different epitopes than those in human breast milk, and consequently they cannot be detected with the microELISAs used, with antibodies specific against recombinant human adhesion molecules. For this reason, it is apparent that the beneficial effects of breast-feeding to the recipient child are lacking, when the newborn infants are formula-fed.
Similarly to previous reports [21][22][23][24][25][26] the results of the present study on slCAM-1, sVCAM-1 and sE-and sLselectin in the serum of healthy term neonates show the following. First, shedding of slCAM-1, sVCAM-1, sE-and sL-selectin is an established component of the immune system of the newborn infant from birth. Second, the increase of postnatal age has a positive effect on the neonatal serum slCAM-1 and sVCAM-1, suggesting an expansion of the neonatal immune system and response. Breast-feeding seems to be beneficial to this expansion and maturation of the immune system considering the elevated concentrations in human milk of the latter two adhesion molecules. Third, the highly elevated neonatal sVCAM-1 and sE-selectin values, as also reported previously, 24 -26 might be attributed to their strong angiogenic function, 17 considering that in the prenatal period intense angiogenesis is noticed. Finally, in the early neonatal period, sL-selectin values were significantly lower then in healthy adults, due possibly to the well-known functional immaturity of neonatal leucocytes, characterized by diminished expression of L-selectin and impaired shedding of sLselectin. 27,28 The finding in the present study of significantly higher slCAM-1 and sL-selectin values in neonates exclusively breast-fed than in neonates supplemented also with cow's-milk formula, suggests that breastfeeding, at least in the very early neonatal period, offers the recipient newborn infant what it is lacking, since these two molecules show lower neonatal values than those in healthy controls.
In conclusion, the findings of this study reconfirm the presence of slCAM-1, sVCAM-1 and sE-selectin, reveal that of sL-selectin in human milk in the early lactation period and indicate that all four adhesion molecules are physiologically important constituents of human breast milk in the very early postpartum period. This suggests potential immunomodulatory and anti-inflammatory effects of breast-feeding on the recipient newborn infant, as well as the promotion of maturation, development and expansion of the neonatal immune systems. Only slCAM-1 in human milk seems to derive, at least partially, from the maternal circulation, while for the origin of the remaining three adhesion molecules only putative suggestions may be proposed.