Superoxide Anion Generation in Human Milk Macrophages: Opsonin-Dependent Versus Opsonin-Independent Stimulation Compared with Blood Monocytes

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Abstract
Macrophages are believed to play an important role within the immunoprotective effects of human breast milk. It was the purpose of this study to evaluate the capability of human milk macrophages (MMPhi) to generate superoxide anions (O2(-)) in comparison with peripheral blood monocytes (BMo) after stimulation with opsonized and unopsonized zymosan. Potential inhibitors of attachment and phagocytosis such as mannose and cytochalasin B were used. Expression of the mannose receptor on MMPhi was demonstrated by staining with MAb. BMo generated more O2(-) than MMPhi (417 +/- 79 versus 216 +/- 15 nmol O2(-)/mg protein, p < 0.05) after stimulation with opsonized zymosan. When unopsonized zymosan was used as a serum-independent stimulus, BMo generated slightly less O2(-) in comparison with MMPhi (150 +/- 34 versus 176 +/- 18 nmol O2(-)/mg protein, p < 0.05). These findings imply a higher proportion of opsonin-independent phagocytosis in MMPhi than in BMo (82 versus 36 %). Preincubation with mannose resulted in a significantly higher reduction of O2(-) generation in MMPhi compared with BMo stimulated with opsonized zymosan, whereas no difference was found when unopsonized zymosan was used. After addition of cytochalasin B, equal inhibition of O2(-) generation was observed regardless of the cell type or stimulus used. Thus, MMPhi are stimulated to a greater extent by serum-independent mechanisms than BMo. As opsonins like complement or IgG are rare in the colostrum and the neonatal intestinal environment, such a differentiation toward serum-independent phagocytic abilities could play an important role for protective functions of human MMPhi. Possible involvement of the mannose receptor and the beta-glucan receptor in this specialization are discussed.