Breast milk macrophages spontaneously produce granulocyte-macrophage colony-stimulating factor and differentiate into dendritic cells in the presence of exogenous interleukin-4 alone

Abstract

Peripheral blood monocytes extravasate and differentiate into tissue macrophages to mediate effective local defence, but how tissue-specific stimuli and environments may influence their functions remains unknown. Here, we found that peripheral blood monocytes gained the ability to produce granulocyte-macrophage colony-stimulating factor (GM-CSF) upon exposure to breast milk and differentiated into CD1+ dendritic cells (DCs) in the presence of exogenous interleukin-4 (IL-4) alone. This in vitro observation appeared physiologically relevant since macrophages that were freshly isolated from breast milk were also found to produce GM-CSF spontaneously. Furthermore, in contrast to peripheral blood monocytes that differentiated into DCs only in the presence of both exogenous GM-CSF and IL-4, differentiation of breast milk macrophages into DCs was induced by incubation with exogenous IL-4 alone. These IL-4-stimulated breast milk macrophages were efficient in stimulating T cells, suggesting their potential role in mediating T-cell-dependent immune responses in situ. On the other hand, unexpected expression of DC-SIGN, a DC-specific receptor for human immunodeficiency virus (HIV), even in unstimulated breast milk macrophages, may favour HIV infection, resulting in an increased risk of mother-to-infant vertical transmission of the virus via breast milk. Thus, tissue-specific development of macrophages is often linked to effective local immunity, but may potentially provide an opportunity for a pathogen to spread and transmit.

Figure 1

BMMφ were morphologically distinct from their possible precursor PBMo. BMMφ and PBMo (inset) were subjected to May–Grünwald staining, and viewed under a light microscope. Note that BMMφ contained numerous inclusions (arrows) in their large cytoplasm.

Figure 2

BrMMφ were phenotypically distinct from PBMo. (a) Purified PBMo (upper panels) and BrMMφ (lower panels) were analysed for surface expression of indicated proteins by flow cytometry. Specific staining (filled area) and negative control staining (open area) were shown in each panel. (b) PBMo and BrMMφ were analysed for mRNA expression of CD83 (upper panel) and GAPDH (lower panel) by RT-PCR. Note that a specific band representing CD83 mRNA expression (arrow) was detected only for BrMMφ, but not for PBMo, whereas both cell types expressed the house-keeping gene, GAPDH.

Figure 3

BrMMφ, but not PBMo, spontaneously produced GM-CSF. (a) Freshly isolated PBMo from six subjects and BrMMφ from 13 subjects were cultured separately for 1 day, and the amount of GM-CSF released into the medium was determined by ELISA and plotted. Note that, whereas no PBMo preparations secreted detectable levels of the cytokine, BrMMφ isolated from most donors produced significant levels of GM-CSF (P < 0·0035; Mann–Whitney U-test). (b) GM-CSF expression in PBMo and BrMMφ was also examined at the transcriptional level by RT-PCR. All the BrMMφ preparations from seven different donors were found to express the GM-CSF gene (arrow) whereas none of the five PBMo preparations expressed the gene.

Figure 4

GM-CSF was produced from PBMo after phagocytosis of breast milk. Purified PBMo were cultured with either media alone, 25% breast milk supernatant, or 0·1% latex beads. After 4 hr, the cells were washed, and then cultured for an additional 30 hr. At the end of the culture, the culture supernatants were collected and GM-CSF concentration was determined by enzyme-linked immunosorbent assay. Light microscopic observation of latex-bead-treated cells revealed that over 90% of cells contained phagocytosed beads intracellularly (not shown).

Figure 5

BrMMφ differentiated into CD1⁺ dendritic cells with high T-cell stimulatory activity after incubation with IL-4 alone. (a)Surface expression of CD1a, CD1b and CD14 molecules on cytokine-activated PBMo and BrMMφ and mock-treated BrMMφ was determined by flow cytometry. Broken lines represent negative staining. Mock-treated PBMo were not analysed due to their poor viability. (b) IL-4-stimulated BrMMφ developed plasma membrane projections (arrows in right panel), which were not apparent in BrMMφ cultured without IL-4 (left panel). Cells were subjected to May–Grünwald staining, and viewed under a light microscope. (c)To assess the ability of cytokine-stimulated BrMMφ to activate naïve T cells, allogeneic cord blood T cells (1 × 10⁴/well) were cultured for 5 days with the indicated numbers of irradiated BrMMφ that were preincubated with either GM-CSF plus IL-4, IL-4 alone, or medium alone, and the [³H]thymidine uptake during the last 12 hr of culture was measured. Results were expressed as counts per min ± SEM.

Figure 6

Freshly isolated PBMo and BrMMφ as well as IL-4-stimulated BrMMφ were analysed (a)for mRNA expression of DC-SIGN (upper panel) and GAPDH (lower panel) by RT-PCR and (b) for protein expression of DC-SIGN by FACScan. In FACScan analysis, specific staining (filled area) and negative control staining (open area) were shown in each panel. Note that fresh BrMMφ, but not PBMo, expresses the DC-SIGN gene and protein and its expression was up-regulated after IL-4 stimulation.