Superoxide Anion Generation in Human Milk Macrophages: Opsonin-Dependent Versus Opsonin-Independent Stimulation Compared with Blood Monocytes
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0031-3998/01/4903-0435 PEDIATRIC RESEARCH Vol. 49, No. 3, 2001 Copyright © 2001 International Pediatric Research Foundation, Inc. Printed in U.S.A. Superoxide Anion Generation in Human Milk Macrophages: Opsonin-Dependent Versus Opsonin-Independent Stimulation Compared with Blood Monocytes RÜDIGER ADAM, FRANK KUCZERA, HENRIK KÖHLER, AND HORST SCHROTEN Zentrum für Kinderheilkunde, Heinrich-Heine-Universität, 40225 Düsseldorf, Germany ABSTRACT Macrophages are believed to play an important role within the found when unopsonized zymosan was used. After addition of - immunoprotective effects of human breast milk. It was the cytochalasin B, equal inhibition of O2 generation was observed purpose of this study to evaluate the capability of human milk regardless of the cell type or stimulus used. Thus, MM⌽ are ⌽ - macrophages (MM ) to generate superoxide anions (O2 )in stimulated to a greater extent by serum-independent mechanisms comparison with peripheral blood monocytes (BMo) after stim- than BMo. As opsonins like complement or IgG are rare in the ulation with opsonized and unopsonized zymosan. Potential in- colostrum and the neonatal intestinal environment, such a differ- hibitors of attachment and phagocytosis such as mannose and entiation toward serum-independent phagocytic abilities could cytochalasin B were used. Expression of the mannose receptor on play an important role for protective functions of human MM⌽. MM⌽ was demonstrated by staining with MAb. BMo generated Possible involvement of the mannose receptor and the -glucan - ⌽ Ϯ Ϯ - more O2 than MM (417 79 versus 216 15 nmol O2 /mg receptor in this specialization are discussed. (Pediatr Res 49: protein, p Ͻ 0.05) after stimulation with opsonized zymosan. 435–439, 2001) When unopsonized zymosan was used as a serum-independent - stimulus, BMo generated slightly less O2 in comparison with ⌽ Ϯ Ϯ - Ͻ MM (150 34 versus 176 18 nmol O2 /mg protein, p Abbreviations 0.05). These findings imply a higher proportion of opsonin- MM⌽, milk macrophages independent phagocytosis in MM⌽ than in BMo (82 versus BMo, blood monocytes - -1 -1 - -1 -1 36%). Preincubation with mannose resulted in a significantly nmol O2 ·mg p ·30' , nmol O2 ·mg protein ·30 min - ⌽ higher reduction of O2 generation in MM compared with BMo MR, mannose receptor stimulated with opsonized zymosan, whereas no difference was CR3, complement receptor type 3 Besides well-known nutritional benefits, transfer of antimi- different subclasses of IgG, IgA, and C3b receptors and syn- crobial activity is of great importance in infant breast-feeding. thesize various humoral defensive factors such as complement A great variety of humoral defense factors (e.g. secretory Ig, factors, lactoferrin, and lysozyme (1–5). especially sIgA, lactoferrin, lysozyme, oligosaccharides, mu- Because human MM⌽ remain viable in conditions similar to cins, and others) contribute to the beneficial effects. In addition, those in the small intestine (6), show relative resistance to an large numbers of viable cells are present in human colostrum environment with a pH Ͻ3, and resist trypsinization (7), it seems and breast milk with a high proportion of macrophages likely very likely that MM⌽ can develop their immunoprotective func- being responsible for antiinfectious properties. tions within the gastrointestinal tract of the breast-fed baby. These human MM⌽ with a diameter of 18 to 40 m contain Interaction of macrophage membrane receptors with com- a high amount of phagocytosed lipids, “foamy cells.” Their plementary coating substances on a pathogen’s surface is well morphologic and cytochemical properties are similar to differ- described as opsonophagocytosis. These coatings (opsonins), entiated macrophages. For example, they bear Fc receptors for derived from various sites of the hosts immune system, consist of bridging serum factors such as Ig, the iC3b fragment of C3 complement factor, C-reactive protein, surfactant proteins A Received February 29, 2000; accepted September 22, 2000. Correspondence: Horst Schroten, M.D., Zentrum für Kinderheilkunde, Heinrich-Heine- and D, and the mannose-binding protein (8). Universität, Düsseldorf, Moorenstraße 5, 40225 Düsseldorf, Germany. Interaction of carbohydrate-binding proteins termed lectins Presented in part at the 17th Scientific Congress of the European Society for Pediatric Hematology and Immunology, Washington, D.C., U.S.A., May 11–15, 1999. with complementary carbohydrate chains is another primary Published in abstract form in Pediatric Research 45:744, 1999. mechanism of mediating attachment of pathogens. This phago- 435 436 ADAM ET AL. cytic process, known as serum independent or lectinophago- ethylmaleimide stopped the induced oxidative metabolism af- cytosis, is promoted, for example, through the MR or a lectin ter treatment with the stimulants for 30 min. Reduction of site on the CD11b/CD18 (MAC-1) integrin, the -glucan cytochrome c was quantified spectrophotometrically (550 nm). receptor (8). The assay was repeated with superoxide dismutase to correct After recognition and attachment of pathogenetic particles, for oxygen-independent reduction of cytochrome c. Results - the process of phagocytosis progresses with different patterns were expressed in nmol O2 /mg protein. of internalization and mostly culminates in the triggering of the respiratory burst with the release of microbicidal oxygen me- Stimulating Agents tabolites. It is hypothesized that due to a lower availability of opsonizing factors in the milk (9) and the gastrointestinal tract Opsonized and unopsonized zymosan (Sigma Chemical Co., of the infant, human MM⌽ are activated by other mechanisms St. Louis, MO, U.S.A.) was used as a stimulus. Serum from than BMo. healthy donors was taken for opsonization. The zymosan par- Therefore, this study was undertaken to investigate opsono- ticles were autoclaved for 30 min, washed twice with PBS, and ϫ 5 and lectinophagocytic properties of human MM⌽ by measur- resuspended in PBS. The samples were adjusted to 2 10 - particles/L and stored at Ϫ70°C until further use. ing the superoxide anion (O2 ) production in comparison with human BMo after stimulation with opsonized and unopsonized zymosan particles. D-Mannose and cytochalasin B as potential Inhibitors inhibitors were used to evaluate attachment and engulfment Mannose and cytochalasin B (Sigma Chemical Co., St. mechanisms. Louis, U.S.A.) were used as potential inhibitors. Two millili- ters of each substance was incubated with the isolated cells for METHODS 15 min at 37°C before the stimulation assay. Mannose was Collection, Preparation, and Culture of Cells prepared in 0.1, 0.25, and 0.5-M solutions, and cytochalasin B was used at a concentration of 1 g/mL. All assays were Human milk was collected from 38 healthy lactating women performed in duplicate. 1–6 d postpartum by hand expression with informed consent at the Department of Gynecology and Obstetrics, Heinrich- Expression of MR Heine-Universität Düsseldorf, Germany, after having obtained approval of the Clinic’s Review Board. The samples were Phagocytes were stained with a monoclonal murine antibody generally obtained before the infants were fed and stored at generated against the human MR (Pharmingen, San Diego, room temperature for up to4hinsterile containers. CA, U.S.A.). BMo and MM⌽ were prepared as described The milk was skimmed by 1:1 dilution with PBS and above and subsequently incubated with the anti-MR antibodies centrifuged at 4°C and 600 ϫ g for 10 min. The pellet was (20 L per pellet) for 10 min, washed with PBS, and centri- resuspended in 25 mL PBS, layered on 25 mL Ficoll-Hypaque fuged at 300 ϫ g for 10 min at 20°C. Staining was evaluated (Pharmacia, Uppsala, Sweden), and centrifuged at 20°C and using a fluorescence microscope (Zeiss Optics, Göttingen, 1000 ϫ g for 20 min. The mononuclear interface cell layer was Germany). washed in PBS and RPMI (GIBCO, NY, U.S.A.) (supplement- ed with penicillin, gentamicin, HEPES, FCS, and glutamine) Statistical Analysis and resuspended in RPMI (with supplements). Purity (Ͼ90%) Statistical analysis was performed by using the paired t test. of the cell preparation was assessed by naphthyl acetate ester- An ␣ error of p Ͻ 0.05 was regarded as significant. ase staining. Cells were enumerated by cell counter (Coulter, Krefeld, Germany) and adjusted at a concentration of 2.5 ϫ RESULTS 105 cells/mL. Monocytic viability of Ն95% was assured by trypan blue exclusion. Two milliliters of the cell suspension Stimulation without Inhibitors were prepared in plastic culture dishes, and cells were allowed - Ϯ to adhere for2hat37°C and 5% CO , then washed vigorously Without any additional stimuli, a O2 production of 40 3 2 - -1 -1 ⌽ Ϯ - with PBS to remove nonadherent cells. nmol O2 ·mg p ·30' in MM and 47 4.7 nmol O2 ·mg -1 -1 Monocytes were isolated from heparinized human blood p ·30' in BMo was measured. After stimulation with opso- Ϯ - from 64 healthy volunteers as described above by Ficoll- nized zymosan, BMo generated 417 79 nmol O2 ·mg -1 -1 ⌽ Ϯ - -1 -1 Hypaque gradient and then treated like the MM⌽. p ·30' , and MM generated 216 15 nmol O2 ·mg p ·30' . Ϯ The protein content in the samples was determined accord- When unopsonized zymosan was used, BMo produced 150 - -1 -1 ⌽ Ϯ ing to the method of Lowry et al. (10). Only samples with a 35 nmol O2 ·mg p ·30' , and MM generated 176 18 nmol - -1 -1 protein content between 40 and 100 g were used (11). The O2 ·mg p ·30' . Thus, peripheral BMo released a higher ab- - ⌽ cells were stored in PBS on ice (maximum, 15 min) until use. solute amount of O2 than human MM when stimulated with - opsonized zymosan, whereas O2 production after activation with unopsonized zymosan demonstrated approximately equal O - Production 2 release of the measured oxygen metabolites. Notably, MM⌽ - - O2 production was measured by reduction of superoxide showed a significantly higher proportion of O2 generation dismutase-sensitive cytochrome c. Addition of 2 mL N- under opsonin-independent stimulation (100 to 82%) than - O2 PRODUCTION IN MILK MACROPHAGES 437 BMo (100 to 36%) in relation to opsonin-dependent stimula- tion (Fig.