Main

Human milk contains a complex mixture of immunologically active components providing both specific and nonspecific defenses against infectious agents. Among the many bioactive substances detected in breast milk that have immunomodulatory potential are a number of peptide mediators known as cytokines. Proinflammatory cytokines such as TNF-α(1), IL-1β(2), IL-6(3), IL-8(4), and IFN-γ(5), as well as antiinflammatory cytokines, including transforming growth factor-β(6) and IL-10(7), are present in quantities likely to be physiologically significant. Cytokines in human milk may play an immunomodulatory role in the alimentary tract of the recipient infant and potentially affect development of the immune system.

IL-12 has not previously been detected in human milk. This cytokine has been shown to enhance lymphocyte-mediated cytotoxicity(8), T cell proliferation(9), and production of a number of other cytokines, including IFN-γ, TNF-α, granulocyte-macrophage colony-stimulating factor, monocyte-macrophage colony-stimulating factor, and IL-2, -3, and -8(10). IL-12 has also been shown to suppress the synthesis of IgE by B cells in the presence of T cells in vitro(11).

IFN-γ and mononuclear cells, both of which are present in human milk, are associated with the production of IL-12(12), leading us to attempt to determine whether IL-12 may also be present. We report the results of a cross-sectional study investigating the presence of IL-12 in human milk at three stages of lactation, using a specific enzyme-linked immunoassay.

METHODS

Subjects. A single breast milk sample was collected from 39 mothers (ages 18-37 y; parity, 1-5) of healthy term infants who were attending a research clinic at Flinders Medical Centre (FMC), Adelaide, Australia. Of these women, 31 (80%) had delivered vaginally, and 11 (29%) smoked cigarettes. Data relating to maternal atopic status (allergic rhinitis, asthma, dermatitis/eczema, or food allergy) and any illness or infection suffered by the mother during the week before sample collection were recorded. Of the 38 women from whom data were collected, 11 (28%) reported some form of illness or infection during the week preceding sample collection, and 24 (63%) reported some degree of atopic history. The study was approved by the Clinical Investigations (Ethics) Committee of FMC, and informed consent was obtained before enrollment.

Protocol. After manual expression into sterile polypropylene containers, milk samples (2-5 mL) were categorized according to stage of lactation: colostrum (≤6 d, n = 13), transitional (11-16 d, n = 13), or mature (40-46 d, n = 13). All samples were stored at 4°C and were processed within 2 h of collection. The samples were centrifuged at 690 × g for 20 min, and the aqueous phase was stored at -80°C until analysis.

Immunoassay. The IL-12 p70 heterodimer was measured in the aqueous phase of the milk by ELISA, using a p35/p70 primary antibody and p40/p70 detecting antibody combination at concentrations recommended by the manufacturer (Pharmingen, San Diego, CA). The minimum detectable level was 40 pg/mL. There was assumed to be no interference by the p40 IL-12 monomer or a range of other cytokines as reported by the manufacturer.

Data analysis. IL-12 levels obtained are expressed in pg/mL. Samples below the minimum detectable level were assigned a value of 20 pg/mL for statistical analysis. Associations between clinical data and log-transformed cytokine concentration values were examined by Spearman correlation using SPSS for Windows 6.0 (SPSS Inc., Chicago, IL).

RESULTS

IL-12 was detected in 24 of the 39 samples collected. The concentration of IL-12 in all samples with amounts above the minimum level of detection was 1408 ± 2256 pg/mL (mean ± SD; n = 24). The levels of IL-12 found in the aqueous phase of individual samples are shown in Table 1. The median concentration of IL-12 decreased from 310 pg/mL in the colostral samples to 176 pg/mL in the transitional milk samples and was below the minimum detectable concentration in the mature milk samples. No associations were found between maternal illness/infection or atopic profile and IL-12 concentration; however, the large number of samples below the level of detection may make statistical analysis inappropriate.

Table 1 IL-12 (pg/mL) in Aqueous Phase of Human Milk from 39 Mothers
Full size table

DISCUSSION

This is the first report of the presence of IL-12 in human milk. The concentration of IL-12 was found to be within our detection limits in 62% of samples tested. There was a broad scatter of values, regardless of time postpartum, as has been observed for other cytokines detected in human milk(1–7). The wide range of concentrations observed was not readily explained by stage of lactation or maternal clinical characteristics such as recent illness or atopic history.

The presence of IL-12 in the aqueous phase of human milk has previously been investigated with ELISA kits (R&D systems Minneapolis, MN)(13,14). However, IL-12 was not detected, which may reflect the use of antibodies of differing specificity for the antigen of interest. The source of IL-12 in human milk is unknown. However, neutrophils, lymphocytes, and macrophages are found in milk during the first few months of lactation(15), and one or more of these cell types have the potential to secrete IL-12.

The presence of IL-12 in human milk may have broad implications for the recipient infant. IL-12 can stimulate the growth and development of T lymphocytes and natural killer cells(8,9) and, like the classic T helper 1 (Th1) cytokines, IL-2, and IFN-γ(16), IL-12 is involved in the control of Th1/Th2 differentiation(17). The in utero environment of human infants during pregnancy is predominantly regulated by Th2-type cells(18). A supply of exogenous IL-12 from the milk of a breast-feeding mother may assist in addressing the balance between predisposed Th2-type responses and the Th1-type cytokine-driven cell-mediated response.