Dov H. Pluznik

Membrane mobility agent alters the consequences of lectin-cell interaction in a malignant cell membrane.

The membrane mobility agent 2-(2-methoxyethoxy)-ethyl 8-cis-2-n-octyl-cyclopropyl)-octanoate promotes cap formation from wheat germ agglutinin-receptor combinations at the expense of agglutination in membranes of malignant mastocytoma cells.

F20C, a new fluorescent membrane probe, moves more slowly in malignant and mitogen-transformed cell membranes than in normal cell membranes

New fluorescent probes of membrane mobility can be introduced into cell membranes at single points with particles of a membrane mobility agent, A2C. The initial entry of fluorescence from the particle into the cell membrane and the subsequent lateral spread of fluorescence have been observed for cells in suspension. A dramatic difference between the behavior of normal lymphocytes and that of mitogen-transformed and mastocytoma cells is found. Both the initial entry and the spreading of fluorescence are much slower in the transformed and tumor cells than in the normal cells at 18 degrees C. Entry and spread of fluorescence in normal cells become slow enough to be observed only at 12 degrees C or below.

Generation of lipopolysaccharide-induced interferon in spleen cell cultures

Incubation of murine spleen-cell cultures with lipopolysaccharide (LPS) induces interferon (IF) production. Maximal IF levels are obtained after incubation with 100 μg/ml for 10 h. Two inbred mouse strains differing in their ability to generate LPS-induced IF in spleen-cell cultures were used: C3H/eB, which generates high levels of IF (about 60 units/ml), and C3H/HeJ, which fails to generate detectable quantities of IF. In a genetic analysis these strains were hybridized and IF production was determined in spleen-cell cultures from F1 and F2 generations, and from backcrosses of F1 hybrids to parent strains. The results indicate that, in parent strains, a single dominant autosomal gene is responsible for differences in IF production in spleen cultures. LPS-induced IF in spleen-cell cultures resists pH 2 for as long as 48 h, but is labile to heating at 56° C for 30 min. Both macrophages and lymphocytes must be present in cultures for generation of LPS-induced IF. By using mixed cultures of macrophages and lymphocytes from C3H/eB and C3H/HeJ mice, it was shown that macrophages have to interact directly with LPS to enable IF production in the cultures.

Topographic changes of membrane receptors for wheat germ agglutinin during the cell cycle and their relation to cytolysis

Abstract Experiments with synchronous cultures of murine mastocytoma cells indicated that cells incubated with wheat germ agglutinin (WGA) responded with cap formation and cytolysis during the mitotic phase only, but not during the G1, S and G2 phases of the cell cycle. The telophase of mitosis was found to be most sensitive to the cytolytic effect of WGA. Addition of the lectin to cells enhanced movement of the lectin-binding sites to the cleavage furrow during the telophase, as demonstrable by the concentration of fluorescent lectin and microvilli in this region. This phenomenon was followed by osmotic lysis of the cells which could be prevented by addition to the medium of dextran of high molecular weight.

The mechanism of wheat germ agglutinin mediated cytolysis of murine mastocytoma cells.

Abstract The present study was undertaken to test whether cytolysis by wheat germ agglutinin requires lateral mobility of membranal lectin receptor sites into caps. Preincubation of interphase murine mastocytoma cells with 100 μg/ml trypsin promoted cap formation by the agglutinin in about 30% of the cells, followed by cytolysis of these cells. Pretreatment of the cells with NaN 3 , low temperature or glutaraldehyde decreased degree of capping and to some extent decreased the degree of cytolusis, while the addition of antibodies to the agglutinin increased the degree of capping and lysis. A linear relationship with a high correlation coefficient exists between the degree of capping and the degree of cytolysis, suggesting that lateral mobility of membrane wheat germ agglutinin receptors is required for cytolysis by the lectin. Further studies have shown the restricted small hole damage followed by osmotic lysis is responsible for the damage induced by the agglutinin of trypsin-treated mastocytoma cells. This was demonstrated (a) by markers of low molecular weight ( 86 Rb) which were released from the cells before those of high molecular weight ( 51 Cr-protein) and (b) by protecting the cells from lysis through incubating them in non-penetrating solutes, such as Dextrans of high molecular weight. It has been calculated the the initial size of the lytic lesion induced by wheat germ agglutinin is ≈ 32 A .

Macrophages as regulators of granulopoiesis.

Much evidence is available which indicates the ability of the macrophage to elaborate factors which can either increase (5, 9, 11, 13, 16, 22) or decrease (17, 20, 21, 25) the proliferation of hemopoietic cells. The question whether committed hemopoietic stem cells proliferation can be regulated by macrophages can now be answered by using the soft agar technique. The introduction of this technique for cloning granulocyte and macrophage precursor cells by Pluznik and Sachs (27) and Bradley and Metcalf (14) provided a new approach to the evaluation of the regulation of granulopoiesis and macrophage formation. The clonal growth in soft agar cultures of such precursor cells, termed colony forming units-culture (CFUC), is wholly dependent on the presence of a stimulatory substance designated colony stimulating factor (CSF) (28). In the mouse assay this substance is produced and/or released by cell suspensions from many organs including hemopoietic tissues, by cell lines and it is also present in the serum and urine (18, 29, 32). White blood cells are a major secretory source of CSF elaborated in vitro. However, the exact relationship between the various white blood cell populations in the production of CSF is unclear. Recently, different independent groups have shown that blood monocytes and tissue macrophages are the major hemopoietic source of CSF in humans and mice (12, 13, 19).

Cellular Requirements for Induction of Colony-Stimulating Factor in Lymphoid Cell Cultures Stimulated by T- and B-Cell Mitogens

The clonal growth of hemopoietic cells in vitro into colonies of granulocytes and/or macrophages requires the presence of a stimulatory substance designated colony-stimulating factor (CSF) (7,21). Data from various clinical and animal experiments suggest that CSF is, or is related to, a physiological regulator of granulopoiesis (review, references 3,12,22,26,27). In the mouse assay, this substance is produced and/or released in vitro by cell suspensions from many organs, including hemopoietic tissue, and by cell lines; it is also present in the serum and urine (12,22,26,27). However, in man, the distribution of this humoral factor is limited mainly to mature leukocytes (12,22,26,27). These observations on the production of CSF by leukocytes led to hypotheses concerning feedback control, whereby mature leukocytes regulate their own production (12,22,26,27).

Endotoxin-Induced Release of Colony-Stimulating Factor

The introduction of an in vitro technique for cloning granulocyte and macrophage precursor cells by Pluznik and Sachs (1965) and Bradley and Metcalf (1966) provided a new approach to the evaluation of the regulation of granulopoiesis and macrophage formation. Committed precursor cells for granulocyte and macrophage differentiation can proliferate in soft agar cultures to form colonies of granulocytes and/or macrophages (Ichikawa et al., 1966). Colonies arise from single cells termed colony-forming cells (CFC) (Pluznik and Sachs, 1966). Colony formation is wholly dependent upon the constant presence of a stimulatory glycoprotein substance named colony-stimulating factor (CSF).

Differential Responses of Macrophage Precursors and Antibody Forming Cells to Immunization

Participation of macrophages in immune responses has been shown in many experiments using various methods (1) (2) (3) (4) (5). It has also been demonstrated that antibody forming cells and their precursors proliferate as a result of injection of antigen (6) (7). As for macrophages and their precursors, this fact has not yet been fully elucidated. The technique of cloning spleen, bone marrow and embryonic liver cells in soft agar (8) (9) (10) now enables quantitative studies of macrophages as well. In this technique certain cells from these hematopoietic organs can multiply in a soft agar medium to which conditioned medium is added (11). This leads to formation of macroscopic cell colonies of four types: macrophages, granulocytes, blast cells, and a mixture of macrophages and granulocytes (9) (12). It has been shown that macrophage colonies originate from a single precursor cell (11). Therefore each macrophage colony in the soft agar represents a precursor cell.