Milk Macrophages Research Articles

Ultrastructural localization of IgE in peripheral blood monocytes from atopics.

Monocyte-enriched preparations derived from peripheral blood leukocytes of atopics were probed via a cocktail comprising peroxidase-conjugated (Fab1)2 fragments of two monoclonal antibodies against human IgE. Reaction product indicative of intracellular IgE was identified by electron microscopy in both large and small vacuoles, and at high magnification exhibited a characteristic granular deposition pattern consistent with highly concentrated (perhaps insolublized) material. IgE-containing vacuoles were observed with comparable frequency to those containing IgG, despite the greater than 10,000-fold relative excess of the latter in serum suggesting highly selective uptake of IgE by the monocytes. These results are similar to those reported recently for IgA in human milk macrophages.

Lactoferrin inhibits prostaglandin E2 secretion by breast milk macrophages.

The interaction between human breast milk macrophages and lactoferrin (LF) in its native form was studied in in vitro culture. Competitive inhibition-binding studies with 125I-LF and unlabeled LF showed that a specific receptor for LF was present on breast milk macrophages. LF at concentrations of 10(-6) -10(-9)M resulted in a dose-dependent inhibition of prostaglandin E2 secretion by breast milk macrophages (control-45 +/- 7; LF 10(-6)M -9 +/- 1 ng/ml/10(6) cells). This inhibitory effect was also observed when the macrophages were stimulated with Concanavalin A (LF 10(-6) M -80 +/- 5; 10(-9) M -45 +/- 8% inhibition of prostaglandin E2 secretion by Concanavalin A stimulated macrophages). Lactalbumin and lactoglobulin had no effect. Similar concentrations of LF had no effect on lysozyme production. We also demonstrated that human milk macrophages are capable of eliciting an oxidative burst as measured by superoxide or hydrogen peroxide production when stimulated by phorbol myristate acetate in in vitro culture. (basal superoxide -1.4 +/- 0.3; phorbol myristate acetate 28.8 +/- 3.5 nmol/1 X 10(6) cells/90 min; basal hydrogen peroxide 11.7 +/- 4.6; phorbol myristate acetate -57.5 +/- 2.3 nmol/mg protein/90 min). LF had no effect on the oxidative burst. These results suggest that interaction of aqueous and cellular components of breast milk may occur and result in varied physiological effects.

Defective chemotactic response of human alveolar and colostral macrophages

This report describes the chemotactic response of human alveolar macrophages (AMφ) and milk macrophages (MMφ) to a panel of chemotactic agents: endotoxin (EAS) and zymosan (ZAS) activated serum, lymphocyte derived chemotactic factor (LDCF) and formylated synthetic peptides. The locomotion studies were compared with the responses of peripheral blood monocytes. Both AMφ and MMφ exhibited an extremely poor chemotactic response to all agents in comparison with the monocytic response. When monocytes were cultured for long periods, a defective response was likewise demonstrated. The chemotactic response was significantly higher in AMφ from smokers. The stimulated locomotion was not increased by the addition of a surfactant lipoprotein to the AMφ suspension. Moreover, monocytes incubated with fat- and cell-free human milk exhibited lower chemotactic responses than normal monocytes and practically in the same range as that obtained with MMφ.

Phagocytosis-associated oxidative metabolism in human milk macrophages.

Evidence exists that human milk macrophages, which are the most abundant cells in milk, play an important role in the protection of the newborn infant against infection. We investigated the oxidative metabolism of milk macrophages by measuring luminol-dependent chemiluminescence, generation of superoxide anion (O2-) and production of hydrogen peroxide (H2O2) in the resting state and after stimulation with opsonized zymosan or phorbol myristate acetate (PMA). Generally, macrophages generated luminol-dependent luminescence, O2- and H2O2 with either stimulus to a similar extent as did blood monocytes. In addition, macrophages killed Escherichia coli and Staphylococcus aureus as effectively as did blood monocytes (approximately 75%, 120 min). Acidification of macrophages in milk (pH 1, 30 min) only slightly reduced their PMA-stimulated production of oxygen radicals. Bacterial killing by macrophages preincubated at pH 1 was about 70% that from controls maintained at pH 7. When macrophages were cultured for several days in endotoxin-free medium, their ability to produce oxygen metabolites declined. By continuous treatment with bacterial LPS (10 ng/ml), milk-macrophages could be "primed" to release large amounts of oxygen intermediates. In addition, the O2- response of macrophages cultured without bacterial products could be partially restored by the addition of LPS to the culture. We conclude that milk macrophages are capable of releasing large amounts of oxygen metabolites, and could contribute to the protection of neonates against bacterial and fungal infections.

Prostaglandin E2 and plasminogen activators in human milk and their secretion by milk macrophages.

Human milk was shown to contain prostaglandin E2 (PGE2) and plasminogen activator (PA) at variable concentrations depending on the time of lactation after delivery. Milk PA was functionally and immunologically identical to urokinase. A follow-up study showed that the maximum PGE2 concentrations occurred during the second week while the maximum PA concentration was observed at the end of the first week of lactation. Milk macrophages cultured in vitro were able to secrete both PGE2 and PA. When cells were activated by concanavalin A (ConA) or E. coli lipopolysaccharide (LPS), PGE2 secretion increased dramatically while PA secretion did not. The ability of activated macrophages to secrete PGE2 was at its highest shortly after delivery. It then progressively decreased during lactation. The possible physiological role of PGE2 and PA on the gastrointestinal tract of breast fed infants is discussed.

Decreased response of human milk leukocytes to chemoattractant peptides.

To test the hypothesis that neutrophils and macrophages in human milk may not defend by classical inflammatory mechanisms, experiments were conducted to ascertain whether adherence, orientation, and directed motility of these leukocytes would be enhanced by exposure to chemoattractant peptides including N-formyl-L-methionyl-L-phenylalanine and N-formyl-L-methionyl-L-leucyl-L-phenylalanine and C5a generated from zymosan activated human serum. Adherence and spatial orientation were tested on coverglasses and in Zigmond chambers, and chemotaxis was examined by Boyden chambers and a subagarose technique. Whereas, the adherence, orientation, and directed movement of adult peripheral blood neutrophils and monocytes were significantly enhanced by those chemotactic agents, human milk leukocytes failed to respond. The failure of the response of human milk leukocytes was not due to alterations in maternal peripheral blood leukocytes but appeared to be due partially to inhibitors in human milk. The experiments suggest that human milk leukocytes may be modified in the mammary gland to protect by noninflammatory mechanisms.

A lymphokine regulates expression of alpha-1-proteinase inhibitor in human monocytes and macrophages.

Biosynthesis and secretion of alpha-1-proteinase inhibitor (alpha 1 PI) has been demonstrated in primary cultures of human mononuclear phagocytes, making it possible to study regulation of alpha 1 PI in normal (PiMM) and homozygous-deficient (PiZZ) individuals. In this study, expression of alpha 1 PI by blood monocytes, bronchoalveolar, and breast milk macrophages decreased during 1 wk in culture whereas expression of other secreted proteins increased. The addition of crude supernatants from mitogen-stimulated peripheral blood mononuclear cells to confluent monolayers of mononuclear phagocytes after 1 wk in culture resulted in a 2- to 2.5-fold increase in alpha 1 PI expression. The increase in alpha 1 PI expression was dose- and time-dependent, and involved a mechanism acting at a pretranslational level as shown by an increase in specific messenger RNA content corresponding to the increase in synthesis and secretion of alpha 1 PI. Although alpha 1 PI was expressed in native form and in forms complexed with serine protease by monocytes early in culture, it was expressed in its native form alone when monocytes were incubated with the lymphokine after 1 wk in culture. The regulating factor had the characteristics of a polypeptide and was derived from T lymphocytes, but it was not interferon-alpha, -beta, -gamma, or interleukin 2. This lymphokine also stimulated synthesis of alpha 1 PI in monocytes of homozygous-deficient PiZZ individuals, but had minimal effect on secretion, thereby increasing the intracellular accumulation of the inhibitor and exaggerating the defect in secretion of alpha 1 PI in these individuals. Regulation of mononuclear phagocyte alpha 1 PI expression by a lymphokine provides a model for further analysis of the effect of enhanced synthesis on a defect in posttranslational processing/secretion and for analysis of differential regulation of protease and inhibitor expressed in the same cells.

Intracellular Immunoglobulins in Human Milk Macrophages

Immunoglobulins (Ig) were localized by immunofluorescence and immunoperoxidase techniques inside human milk macrophages (HMM phi). Immunoelectron microscopy was employed to study the distribution and localization of Ig within HMM phi. IgA and IgM were detected inside phagocytic vacuoles of different sizes in the vicinity of the cellular membrane and in the periphery of larger vacuoles with a dense and homogeneous content. The intramacrophagic IgA represents 5-10% of the total milk IgA. The in vitro release of IgA into the medium was quantitated using an enzyme-linked immunoabsorbent assay. The amount of IgA released was in most samples around 1,720-5,200 ng/10(6) HMM phi. The concentrations of IgM and IgG released by HMM phi were approximately 50-600 and 20-40 ng/10(6) HMM phi, respectively. The concentrations of Ig determined in the lysated pellets of HMM phi ranged between 2 and 100 micrograms/10(6) HMM phi for IgA and around 500 ng/10(6) HMM phi for IgM. Increased IgA release occurred when HMM phi were cultured in medium with a pH lower than 3. IgA concentrations were not very different between pH 5 and pH 7 and alkalinization of the medium did not produce any significant effect on IgA release. Release of IgA and IgM was not significantly increased when HMM phi were cultured in the presence of phorbol myristate acetate, formylated peptides, latex particles, lymphocyte-derived chemotactic factor or cholate. The kinetic study of IgA release by HMM phi, both with or without adding these stimuli to in vitro culture, indicates that IgA release depends on time and that these substances do not have any effect on it.

Immunohistologic analysis of a human pulmonary alveolar macrophage antigen

PAM1 is a 200-kDa polypeptide antigen present on lavaged human alveolar macrophages but not on monocytes, peritoneal macrophages, breast milk macrophages, or other normal hematopoietic cells studied by flow cytometry. We have characterized the distribution of expression of this antigen by cells in tissues by using immunohistologic techniques. Normal and diseased lung as well as lymph nodes, spleen, kidney, liver, GI tract, and skin were studied. PAM1 was expressed strongly on the surface and weakly in the cytoplasm of most alveolar macrophages in all 15 of the lung specimens. Occasional interstitial macrophages had weak to moderate staining for this antigen but the majority did not stain. The distribution, pattern, and intensity of staining for PAM1 was the same in normal lung specimens and those with interstitial pneumonitis, despite the increase in mononuclear cells in the latter. Dermal histiocytes and Kuppfer cells expressed PAM1 weakly. Sinus histiocytes in lymph nodes were moderately to strongly positive. Although lymphoid cell suspensions (tonsil) were negative by flow cytometry, five of six lymph nodes had positive cells by immunohistology. PAM1 was also detected on endothelial cells of splenic sinusoids in all 6 specimens but not on any other endothelium. Hence, while PAM1 is expressed most strongly on alveolar macrophages, it can also be demonstrated in other locations using sensitive immunohistologic techniques. Since circulating monocytes are antigen negative and some lung interstitial macrophages bear antigen, PAM1 may be a useful marker for studies of the differentiation of mononuclear cells in the lung.

Oxidative metabolic response and microbicidal activity of human milk macrophages: effect of lipopolysaccharide and muramyl dipeptide.

Mouse macrophages can be primed by exposure in vitro to the bacterial products lipopolysaccharide and muramyl dipeptide (MDP) or in vivo by injection of MDP, so that they produce more of the bactericidal agent superoxide anion (O2-) when stimulated by phagocytosis or by contact with phorbol myristate acetate (PMA). Because little is known about the physiology of human tissue macrophages, we examined release of O2- by milk macrophages obtained from 45 normal women for the ability to undergo priming for greater O2- release. In samples from the same individuals, PMA-stimulated O2- release was similar from colostrum (0 to 3 days postpartum) or from transitional milk (5 to 8 days). Release of O2- by milk macrophages was almost identical to that by blood monocytes from the same women. Milk macrophages phagocytized and killed Candida albicans relatively effectively. Incubation with lipopolysaccharide activated the macrophages in that they were primed for greater PMA-stimulated O2- release. Incubation with the adjuvant MDP or its analog 6-O-(2-tetradecylhexadecanoyl)-MDP did not prime, but incubation with a second analog, 6-O-(stearoyl)-MDP, primed the macrophage for greater O2- release. These results indicated that human tissue macrophages can be primed for greater oxidative response by exposure to bacterial products. Potential exists for the therapeutic use of such immunomodulating agents in the enhancement of host defense.

1033 THE DECREASED RESPONSE OF HUMAN MILK LEUKOCYTES TO CHEMOTACTIC FACTORS

The protective effects of human milk leukocytes (HMLs) may be different from their counterparts in blood. Based upon observations including the limited microbiocidal activity of milk neutrophils and macrophages, we hypothesized that these cells protect by non-inflammatory mechanisms. That possibility was further tested by examining the response of HMLs to well defined chemotactic factors.The adherence, orientation, and chemotaxis of unfractipnated washed HMLs to f-met-leu-phe (10−6−10−9M), f-met-phe (10−6−10−9M) and zymosan activated serum (ZAS) were compared to the responses of unfractionated peripheral blood leukocytes (PBLs). In contrast to PBLs, HMLs adhered poorly to glass, and both adherent and non-adherent HMLs failed to orient toward chemotaxins in Zigmond chambers. In sub-agarose and Boyden chamber studies, no chemotaxis of HMLs was observed, whereas the chemotaxis of PBLs was normal (ie. in subagarose plates: x S.D. with f-met-leu-phe, 1.1 ± 0.2mm; ZAS, 1.1 ± 0.4mm; p<0.01). We then tested whether leukocytes were inhibited by soluble colostral factors. PBLs in cell-free colostrum moved normally under agarose toward f-met-leu-phe or ZAS. Adherence of PBLs in colostrum was significantly decreased; this inhibition however was abrogated by reincubating the PBLs in tissue culture media.Thus, the decreased response of HMLs to common chemotactic agents is further evidence that these leukocytes may be modified to protect by non-inflammatory mechanisms.

Tissue-specific pretranslational regulation of complement production in human mononuclear phagocytes.

The net production of the complement protein C2, C4, and factor B differ among mononuclear phagocytes from peripheral blood and different tissues in experimental animals and in humans. To examine the mechanisms that regulate these differences in humans, the proportions of C2-producing cells, the average single-cell production rate of C2, the posttranslational glycosylation and kinetics of secretion of C2 and factor B, and the amounts of C2 and factor B mRNA were examined in freshly isolated peripheral blood monocytes, monocytes maintained up to 2 wk in culture, and freshly isolated tissue macrophages from breast milk and bronchoalveolar lavage. In addition, the biosynthesis of two other proteins synthesized and secreted by mononuclear phagocytes, C3 and lysozyme, were examined. We report that despite comparable rates of C3 and lysozyme synthesis and similar processing and kinetics of secretion of C2 and factor B, the freshly isolated tissue macrophage differs from the monocyte-derived macrophage in the proportion of C2-producing cells, in the average single-cell production rate of complement, and in the amounts of specific C2 and factor B mRNA. These differences are tissue specific, because C2-specific mRNA content in bronchoalveolar macrophages is considerably greater than in breast milk macrophages, although the amounts of factor B mRNA are comparable. These data suggest that tissue-specific regulation of complement production in human mononuclear phagocytes occurs at a pretranslational level. These studies now provide a basis for investigation of the molecular effects of agents that modulate the biologic functions of monocytes and macrophages.

Expression of the alpha 1-proteinase inhibitor gene in human monocytes and macrophages.

Expression of the alpha 1-proteinase inhibitor (alpha 1PI) gene was studied in human mononuclear cells. Using RNA blot and dot hybridization, alpha 1PI mRNA was detected in human peripheral blood monocytes, bronchoalveolar and breast milk macrophages, but not in B or T lymphocytes. Using incorporation of a radiolabeled amino acid precursor, synthesis and secretion of alpha 1PI were demonstrated in human monocytes and macrophages, but not in lymphocytes. In addition, alpha 1PI was secreted in functionally active form as shown by complexing with serine proteases. Biosynthesis of alpha 1PI by mononuclear phagocytes was greatest during the first 24 hr in culture and progressively decreased over the next 10 days. The reduction in alpha 1PI biosynthesis in vitro involved a mechanism acting at the pretranslational level as alpha 1PI mRNA content also progressively declined over 10 days in culture. The ease of sampling human monocytes and macrophages now permits examination of the biochemical defect in homozygous PiZ and PiS alpha 1PI deficiencies and study of the functional significance of locally produced alpha 1PI in normal tissues and sites of injury or inflammation.

Surface membrane heterogeneity among human mononuclear phagocytes.

Considerable information has been accumulated about the functional and metabolic differences that exist among human mononuclear phagocytes isolated from different anatomic sites. This heterogeneity may reflect the influence of environmental factors that stimulate circulating monocytes to differentiate in a distinctive manner, or may suggest the existence of predetermined subsets of macrophage precursors. The present study was designed to evaluate macrophage diversity at the membrane level by defining the expression of antigenic determinants on the surface of these cells. The expression of two new membrane differentiation antigens, BMM1 and PAM1, and of Mo1 and Mo2 were evaluated on blood monocytes, macrophages derived from cultured monocytes, breast milk macrophages (BMM), pulmonary alveolar macrophages (PAM), and peritoneal macrophages. Anti-BMM1 is a mouse IgG2a monoclonal antibody obtained by immunization with breast milk cells that recognizes an antigen expressed only on BMM but not on blood monocytes (or any other circulating cells), PAM, and peritoneal macrophages. BMM1 expression is also seen on HL60 promyelocytes induced toward macrophage differentiation by lymphokines but not by phorbol diester. Anti-PAM1, an IgG1 monoclonal antibody raised by immunization with PAM, reacts with an antigen found only on PAM. Blood monocytes, BMM, and peritoneal macrophages all share the previously described differentiation antigens, Mo1 and Mo2 (as well as Ia), whereas these markers are not found on PAM. BMM1 is associated with a two subunit surface protein of 46 and 40 Kd, and PAM1 resides on a 200 Kd polypeptide. The appearance of new antigens during differentiation (BMM1 on milk macrophages and PAM1 on PAM) and the disappearance of antigens more common to the macrophage lineage (absence of Mo1 and Mo2 on alveolar macrophages) indicate a significant heterogeneity at the membrane level among phagocytes obtained from different anatomic sites (but arising from a common myeloid precursor). The functional relevance of these structures selectively expressed by BMM and PAM is the object of further investigation.

Phenotypic characterization and functional activity of human milk macrophages

A large population (about 80%) of the cells obtained from colostrum and early human milk were considered to be macrophages by the following criteria: nonspecific esterase stain, adherence, phagocytosis and IgG-Fc receptor expression. The majority of freshly isolated human milk macrophages (HMMΦ) stain for the monocyte antigen OKM1. Another monocyte antigen, 61D3, was expressed only by 30% of HMMΦ. Class II antigens were expressed by HMMΦ. About 85% of the cells were DR-positive whereas 50% were DS-positive as assessed with a panel of monoclonal antibodies directed against class II antigens. Monocyte and class II antigens were gradually lost during in vitro culture. HMMΦ can support proliferative response to antigens and mitogens when cocultured with autologous peripheral T cells. The proliferative response was significantly reduced when monoclonal antibodies to DR or DS were added to the assay. These results indicate that HMMΦ have the phenotype and functional characteristics of antigen presenting cells.

Studies on human milk macrophages: effect of activation on phagocytosis and secretion of prostaglandin E2 and lysozyme.

Breast milk macrophages cultured in vitro synthesized and secreted increasing amounts of protein, lysozyme, and prostaglandin E2(PGE2) into the extracellular medium. These cells were also shown to actively phagocytose labeled zymosan particles in culture. Morphologic characteristics, phagocytosis, and secretory responses of the macrophages were altered depending on the presence of various stimuli in the culture. Concanavalin A, endotoxin and zymosan particles, but not latex particles, all resulted in an increased PGE2 secretion into the medium. Although total protein synthesis was not altered by any of these stimuli, Concanavalin A and endotoxin resulted in a decreased lysozyme concentration in the extracellular medium. Concanavalin A enhanced, whereas endotoxin and prior phagocytosis of latex particles inhibited phagocytosis of labeled zymosan particles. These findings indicate that phagocytosis and secretions of milk macrophages may be altered depending on the nature of the stimulating agent.

A study of cells in the mammary secretions of sows

The morphology and some of the in vitro functional properties of the cells in the mammary secretions of sows have been examined. A mean cell yield of 1 × 10 7 cells/ml was obtained from sow colostrum but during the first week post-partum the yield decreased approximately 10 fold. The polymorphonuclear leucocyte was the predominant cell type in colostrum and milk and was associated with varying proportions of lymphocytes, macrophages and epithelial cells. The phagocytes of sow milk ingested heat-killed yeast, although the phagocytic index for milk macrophages was low compared with autologous neutrophils and alveolar macrophages. Milk whey provided an effective opsonising medium for yeast ingestion. Intra-mammary immunisation of sows with ovalbumin induced antigen-reactive lymphocytes in both peripheral blood and milk.

Inhibition of natural killer activity by human bronchoalveolar macrophages.

Mononuclear phagocytes were isolated by adherence from peripheral blood, peritoneal exudates, early lactation milk, ovarian carcinomatous ascites and bronchoalveolar lavages. Their capacity to modulate natural killer (NK) activity was assessed by mixing them with blood lymphocytes and by measuring lysis of 51Cr-labeled K562 cells. Unlike other mononuclear phagocyte populations, alveolar macrophages caused a marked dose-dependent inhibition of NK activity. Significant inhibition (40%) of the expression of cytotoxicity was evident at a ratio of alveolar macrophages to lymphoid cells of 0.12:1, and more than 80% suppression was usually observed at a ratio of 0.5:1. Blood monocytes, peritoneal and milk macrophages were consistently inactive up to the highest ratio tested, 2:1. Inhibition of the expression of NK activity by alveolar macrophages was observed at lymphocyte to K562 ratios ranging from 6:1 to 100:1 and over a 4 h or 20 h 51Cr release assay. Alveolar macrophages also inhibited interferon-stimulated cytotoxicity. Alveolar macrophages are unique among the mononuclear phagocyte populations studied in their capacity to inhibit the expression of NK activity effectively, and they could play a role in determining the low levels of NK activity associated with human pulmonary tissue.

Human milk banking. Effect of refrigeration on cellular components.

The storage of human milk at 4 C for 48 hours after expression resulted in a significant (p < 0.02) loss of cellular viability. Further, the concentration of milk macrophages and neutrophils decreased significantly (p < 0.02), presumably via cell adhesion to the milk container or cytolysis. The milk lymphocyte concentration, however, was not significantly affected by storage. Milk passage through a nasogastric feeding catheter had no additional effect on cell viability or concentration. Thus, while current banking methodologies allow greater availability of breast milk's resistance factors to newborn infants, they do so with significant, but not serious, alterations in both the quantity and quality of these components.

The Milk Mononuclear Phagocyte

A large number of mononuclear phagocytes are present in human colostrum and milk and are transferred from mother to baby. Although one can only speculate on their role in vivo, there is a modest amount of information about their characteristics in vitro which will be reviewed in this paper. About 80% of the one to two million leukocytes in colostrum and early human milk are mononuclear phagocytes1,2 as determined by esterase staining,3 latex ingestion, and glass adherence. While See Table in the PDF Document their concentration diminishes with longer lactation, the number of macrophages secreted daily is large (Table 1). The milk mononuclear phagocyte spreads avidly onto glass surfaces extending long filamentous processes into the environment (Fig 1). Trypan blue is rapidly taken up into the lysozomes of living cells (Fig 2) as is acridine orange. These cells are lipid laden (Fig 3) and on ultrastructural analysis one sees that the lipid inclusions are not always membrane bound (Fig 4). Mitochondria, an indication of oxidative metabolism, and rough and smooth endoplasmic reticulum, suggesting active protein synthesis, are plentiful. Comparisons between milk and blood mononuclear phagocytes are listed in Table 2. Lysozyme is one of the proteins synthesized and secreted by the milk mononuclear phagocyte. By competitive binding, radioimmunoassay milk macrophages can be shown to release 100 ± 8 ng lysozyme/ 5 x 105 cells into the supernatant; the level is reduced to 20 ± 4 ng when these cells are cultured in the presence of 10 µg/ml of cyclohexamide, an inhibitor of protein synthesis. Moreover, it appears that these cells make C3 and C4.