R. de Water

Heterogeneity in wheat germ agglutinin binding by mouse peritoneal macrophages.

SummaryThe binding of the lectin wheat germ agglutinin (WGA) to the cell surface of monocytes and macrophages obtained from the stimulated peritoneal cavity of mice was investigated electron microscopically, using ovomucoid-gold as an indirect marker. Resident (tissue) macrophages, identified by the presence of PO activity in the rough endoplasmic reticulum as well as in the nuclear envelope, showed low WGA binding, whereas monocytes and monocyte-derived macrophages with PO activity in the granules showed high WGA binding. Since cells devoid of PO activity showed variable WGA binding, the value of this gold-WGA-binding technique for discrimination on a quantitative basis between resident macrophages and monocytes or monocyte-derived macrophages, is discussed.

A cytochemical method for the demonstration of 5′-nucleotidase in mouse peritoneal macrophages, with cerium ions used as trapping agent

SummaryA method was developed for the demonstration of 5′-nucleotidase in murine peritoneal resident macrophages. The cells are incubated cytochemically without agitation and cerium chloride is used as a trapping agent. Under these conditions, the great majority of the macrophages in the unstimulated peritoneal cavity show enzyme activity in the plasma membrane. In the presence of AMP-S (an AMP analogue inhibiting 5′-nucleotidase, as shown biochemically) there was a decrease in both the number of positive macrophages and the amount of reaction product on the plasma membranes. This indicates that the enzyme activity detected by our cytochemical procedure is attributable to 5′-nucleotidase.

Ultrastructural localization of the circulating cathodic antigen in the digestive tract of various life-cycle stages ofSchistosoma mansoni

In the present study the ultrastructural localization of the schistosome-derived circulating cathodic antigen (CCA) within the digestive tract of various life-cycle stages of the blood flukeSchistosoma mansoni has been determined. Use was made of CCA-specific monoclonal antibodies in a two-step gold-labeling procedure. The following results were obtained: In cercariae gold label was found in the cytoplasm and in the luminal surface coat of the gut epithelium. The oesophagus of this life-cycle stage of the parasite showed minimal CCA reactivity, as gold label was limited to the luminal surface coat, locating proximally to the gut. In 3 1/2-week-old worms and in adult male and female worms CCA was demonstrable in the Golgi apparatus, in cytoplasmic vesicles and in the luminal surface coat; in the caudal quarter of the gut of adult worms CCA was also present in lysosome-like bodies. The oesophagus of these worm preparations did not show any CCA reactivity. In miracidia CCA was not present.

Automated measurement of immunogalactosidase reactions with a fluorogenic substrate by the aperture defined microvolume measurement method and its potential application to Schistosoma mansoni immunodiagnosis.

For automated read-out of immunogalactosidase assays with a fluorogenic substrate, performed in microtitration trays, an inverted fluorescence microscope equipped with a photometer and a scanning stage and operated by a microprocessor has been used. Trays (60 and 96 wells) were scanned in 1 min and corrected measurements and a histogram of the results printed out in a few minutes. A lower level of 4 ng of the reaction product, 4-methylumbelliferone, was detected. By making the measuring system flexible we were also able to use it for fluorescence measurements of individual agarose beads or cells and for extinction measurements in ELISA reactions with a chromogenic substrate. On comparing FITC and galactosidase conjugates in a DASS-test, and peroxidase and galactosidase conjugates in an ELISA, with Schistosoma mansoni infection as test system, it appeared that the galactosidase modification of both assays was 30-50 times more sensitive than the FITC or peroxidase modification. For S. mansoni egg antigen, a lower detectable level of 0.1 ng antigen/ml was achieved.

Heterogeneity of concanavalin A binding by mouse peritoneal macrophages

SummaryThe binding of the lectin concanavalin A (Con A) to the cell surface of monocytes and macrophages collected from the stimulated peritoneal cavity of mice was investigated electron microscopically with horseradish peroxidase-gold as an indirect marker. Individual cells were identified by the cytochemical localization of peroxidatic (PO) activity. In monocytes and monocyte-derived macrophages with PO activity in cytoplasmic granules, the degree of Con A binding was lower than in resident macrophages with PO activity in the rough endoplasmic reticulum and nuclear envelope. An even higher degree of Con A labelling was found on the surface of cells devoid of PO activity. Since the above-mentioned cell populations show a different degree of lectin binding, it is suggested that lectin labelling methods might offer a new tool for quantitative investigation of the differentiation of monocytes and resident macrophages.

Proliferation and differentiation of mononuclear phagocytes in vitro

Mononuclear phagocytes form a cell line originating in the bone-marrow (1). The most immature cell of this line, the monoblast, has been identified and characterized in liquid cultures of mouse bone-marrow (2, 3). This progenitor cell has also been recognized in cultures of human bone-marrow (4). The monoblast is probably not the direct descendent of the pluripotent hematopoietic stem cell, since there is evidence pointing to the existence of other non-adherent precursors between the hematopoietic stem cell and the monoblast (5, 6). In culture, the monoblast has the morphological, cytochemical, and functional characteristics of a mononuclear phagocyte, albeit a primitive one (2, 7–9). Promonocytes (10), which are formed by division of monoblasts, divide and give rise to monocytes. Since monocytes stretch immediately and transform into macrophages, they are not recognizable as monocytes under the light microscope.

Heterogeneity of 5′-nucleotidase activity and wheat-germ agglutinin binding in mononuclear phagocytes

At the electron microscopical level various types of monocytes and macrophages can be recognized after application of a cytochemical method for the demonstration of peroxidatic (PO) activity. PO activity is found in granules of monocytes and exudate macrophages, in the rough endoplasmic reticulum (RER) and nuclear envelope of resident macrophages and in granules as well as in the RER and nuclear envelope of exudate-resident macrophages. PO-negative macrophages lack PO activity (1). All of these cell types occur in the peritoneal cavity after stimulation with various agents and can be generated in bone marrow cultures if the appropriate growth factors are present (2–4).

Expression of 5'Nucleotidase Activity and Wheat-Germ Agglutinin Binding Sites in Mononuclear Phagocytes From Bone Marrow Cultures

The question as to whether the various types of mononuclear phagocyte found in bone marrow cultures and recognized by specific peroxidatic (PO) activity patterns differ in the expression of binding sites for the lectin wheat‐germ agglutinin (WGA) and the activity of the ectoenzyme 5′nucleotidase (5′N) was investigated. Monoblasts, promonocytes, monocytes, and/or exudate macrophages, and exudate‐resident macrophages generally showed a high level of WGA binding, and a considerably lower level was found in the PO‐negative cells and in resident macrophages. 5′N activity was absent in monoblasts, promonocytes, and in the great majority of the monocytes and/or exudate macrophages, but was demonstrable in exudate‐resident macrophages and resident macrophages, as well as in PO‐negative macrophages after 4 days of culture. On the basis of the successive occurrence of the above‐mentioned phenotypes in cultures, the possibility that this diversity in WGA binding and 5′N activity is related to modulation during cell differentiation is discussed. The present findings led to the conclusion that the PO‐negative macrophages, whose origin was previously not entirely certain, are precursors of resident macrophages.

Wheat-germ agglutinin binding in four types of mouse peritoneal macrophage

SummaryAfter stimulation of the mouse peritoneal cavity with newborn calf serum (NBCS), four types of monocyte and macrophage were distinguished on the basis of peroxidase (PO) patterns. These cell types showed heterogeneity in their binding of the lectin wheat-germ agglutinin (WGA). At 16 h after stimulation, monocytes and monocyte-derived macrophages (with PO activity in granules) had a high level of WGA binding; PO-negative macrophages showed moderate WGA binding, and resident macrophages (with PO activity in the RER and nuclear envelope) had low WGA binding. At later time-points after stimulation, each of these cell types lost WGA binding sites. This decrease was related to a process of differentiation and to a modulation, affected by environmental factors. The present results also indicated that PO-negative macrophages can give rise to resident macrophages. Whether these PO-negative cells are monocyte derived or originate otherwise needs further investigation. The fourth type of macrophage, the exudate-resident cell (wtth PO activity both in granules and in the RER and nuclear envelope), with a WGA binding pattern similar to that of monocytes and monocyte-derived macrophages, was considered not to be a resident precursor cell.

Autoradiographical Demonstration of C3b Receptor Activity on Resident Peritoneal Macrophages

SummaryThe present study was performed to evaluate the usefulness of 125I-labelled C3b bound to constituents of sheep erythrocyte membranes (125I-C3b-OR) for the demonstration of C3b receptor activity of resident peritoneal macrophages at the electron-microscopical level. The binding of 125I-C3b-OR to the cells was studied in biochemical and autoradiographical experiments. The amount of cell-associated radioactivity was dependent on the presence of unlabelled aggregated C3b (AC3b) in a dose-response manner, and diminished strongly after functional inactivation of the receptor by trypsin treatment. In addition, it was found that at 4° C most of the label was associated with the cell surface. However, when the incubation temperature was raised from 4° C to 37° C, internalization of the label was observed. These results indicate that 125I-C3b-OR is a suitable agent for further characterization of the C3b receptor-function of resident peritoneal macrophages at the electron-microscopical level.