Scott, Robert E.

Abstract The plasma membrane serves as an important site for the interaction of macrophages and lymphocytes in antigen recognition and of macrophages and foreign agents in inflammatory responses. Despite the critical role of the macrophage membrane in these reactions, studies on the biochemical and functional characteristics of macrophage membranes which need to be performed have been limited by the lack of suitable methods for the isolation of macrophage plasma membranes that maintain functional and antigenic membrane markers. Recently a new method for membrane isolation has been reported which is based on the observation that a variety of sulfhydryl blocking agents can induce the shedding of plasma membrane vesicles of monolayer cell lines. In this paper we demonstrate that guinea pig peritoneal exudate macrophages can also be induced to shed significant quantities of plasma membrane vesicles by such reagents, in particular with a low m.w. aldehyde, and that these vesicles can be isolated by centrifugation. Vesiculation in macrophages is potentiated by disulfide reducing agents, such as dithiothreitol, and by calcium ions. Maximum vesiculation is observed when well spread macrophage monolayers are incubated in a slightly hypertonic vesiculant at pH 7.0 for 2 hr at 37°C. Brief exposure of macrophages to a vesiculant for intervals as short as 30 min is also effective. Macrophage vesicles shed from the cell surface by this procedure have been found to contain receptors for concanavalin A, wheat germ agglutinin, phytohemagglutinin, and lentil lectin. Vesicles also contain Fc and complement (C3) receptors and form rosettes with thymic lymphocytes. It is suggested that macrophage plasma membrane vesicles isolated by this technique should serve as a valuable new system to study the biochemical characteristics of macrophage plasma membranes and their role in immunologic reactions.

ABSTRACT The topography of plasma membrane intramembranous particles in contact-inhibited and transformed cells is a topic of current controversy. We have reported that particles are aggregated in 3T3 cells and that they are randomly distributed in SV3T3 cells. Other investigators have, however, proposed that these differences result from the effect of cryoprotectants and are blocked by aldehyde fixation. The experiments described in this paper re-examine PMP topography in large samples of 3T3 and SV3T3 cells prepared by a variety of preparative methods. The results confirm that 3T3 cells contain aggregated intramembranous particles and that native SV3T3 cells do not, regardless of whether cells are prepared in glycerol, sucrose, tissue culture medium or following prefixation in paraformaldehyde. Data presented in this paper also show that cytochalasin B induces dose -and time-dependent disaggregations of particles in contact-inhibited 3T3 cells. Particle disaggregation results from exposure of cells in situ for 1-3 h to 1-500 ng/ml cytochalasin B. Removal of the drug by washing with reincubation of cells in medium containing serum results in particle reaggregation within 1 to 3 h. These observations suggest that intramembranous particles in 3T3 cells are aggregated in the native state and that membrane-active drugs modulate their distribution by a mechanism possibly involving disruption of linkages between membrane components and the cytoskeleton.

ABSTRACT Plasma membrane vesicles are shed from monolayer cell cultures during incubation in low concentrations of formaldehyde and other sulphydryl blocking reagents. In both 3T3 and SV3T3 cells disulphide reducing agents, including dithiothreitol and mercaptoethanol, potentiate formaldehyde-induced vesiculation. Plasma membrane vesiculation is shown to be a temperature-dependent phenomenon which occurs optimally between 22 and 37 °C and at pH 7·0 to 7·5. Membrane shedding is an energy-dependent phenomenon, requiring monovalent and divalent cations and slightly hypertonic medium. Plasma membrane vesiculation is not affected by pretreatment of cells with inhibitors of protein synthesis, i.e. cycloheximide, nor by agents which disrupt the cytoskeleton.

ABSTRACT 3T3 and SV3T3 mouse embryo cells and a variety of other monolayer cell lines can be induced to form and shed plasma membrane vesicles by exposure to sulphydryl blocking agents including formaldehyde and N-ethyl malemide. Morphological studies show that multiple vesicles are formed and released from individual cells and that the vesicle membrane is continuous with the plasma membrane of the cell. Vesicles measure from 0 ·1 to 15 μm in diameter and are free of detectable contamination with cytoplasmic membranes and organelles. Vesicles also show a 10-fold enrichment in the plasma membrane marker enzyme 5’nucleotidase and are devoid of detectable NADH-cytochrome C reductase and succinic dehydrogenase activity which are marker enzymes for endoplasmic reticulum and mitochondria, respectively. Vesicles have a high cholesterol: phospholipid ratio and show enrichment in sphingomyelin content. They contain receptors for Con A and WGA, approximately 20 size class polypeptides and intra-membranous particles. These results suggest that vesicles are derived from and have the general characteristics of plasma membranes.

Normal human squamous mucosal cells of the soft palate, buccal surface, epiglottis, hypopharynx, floor of the mouth, and tongue were cultured in vitro in serum-free medium. In medium MCDB 153 containing epidermal growth factor, insulin, bovine pituitary extract, and 0.1 or 2.0 mmol/L Ca ** , squamous mucosal cells double every 24 hours. These cells then can be induced to arrest their proliferation reversibly by treatment with transforming growth factor-β or ethionine, and they can irreversibly growth-arrest during senescence or when cultured in growth factor–deficient medium containing 2 mmol/L Ca ** . The latter medium also induces differentiation, as does culture of cells in serum-containing medium. Serum-containing medium furthermore promotes extensive cell stratification and the formation of multilayered squamous mucosal tissue specimens that can be removed intact by Dispase treatment. These specimens represent potential autogenous mucosal grafts that can be used in patients who require reconstructive surgery of the oral cavity and oropharynx. Normal human squamous mucosal cells therefore closely resemble keratinocytes derived from the epidermis in the mechanisms that regulate proliferation and differentiation. This model cell system should facilitate future studies on upper aerodigestive tract squamous mucosal cell physiology and pathophysiology.

Abstract Although osteoblasts contain estrogen receptors, it is unclear whether estrogen has direct effects on osteoblast proliferation and differentiation. We evaluated the effects of 17β-estradiol treatment (1 pM to 10 nM) on the proliferation and differentiation of cultured normal adult human cells that expressed many of the phenotypic characteristics and hormonal sensitivities of mature osteoblasts (hOB cells). Treatment of hOB cells with estradiol for as long as 144 h did not affect the rate of DNA synthesis and had minimal, if any, effects on differentiated function. Whereas alkaline phosphatase activity was increased by nearly twofold (P < 0.01) when the hOB cells were treated with 1 nM 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], treatment with estradiol had no effect when given alone and did not affect the cells' response to 1,25-(OH)2D3. Similarly, the release of bone gla protein (BGP, osteocalcin) was induced by treatment with 1,25-(OH)2D3 (P < 0.05), but estradiol treatment did not affect this response. Cellular levels of mRNA for alkaline phosphatase and BGP were not altered by estradiol treatment. We conclude that estradiol treatment does not have major effects on the growth or differentiation of cultured hOB cells. These results are consistent with previous observations in vivo that indicate estrogen acts principally to decrease bone resorption, not to modulate its formation.

Abstract A new bone cell line was established by transfecting normal adult human osteoblast-like (hOB) cells, derived from a 68-year-old woman, with the plasmid pSV3 neo. The plasmid included coding sequences and promotors for the large and small T antigens of the SV40 virus as well as resistance to the antibiotics neomycin and G418. A single antibiotic-resistant colony was located and cloned. Large tumor antigen production in the clonal cell line was confirmed by indirect immunofluorescence study. Treatment with 1,25-dihydroxy-vitamin D3 increased steady-state concentrations of protein and mRNA for osteocalcin and for alkaline phosphatase. Northern blot analyses also demonstrated the presence of mRNAs for α(I)-procollagen, osteopontin 1a, transforming growth factor β, and interleukin-1β. The plasma membrane calcium pump and osteonectin were identified by immunocytochemical analysis. These cells produced a matrix that mineralized when β-glycerophosphate was added to their cultures. As assessed by functional receptor assays, both estrogen and androgen receptors were present and functional, although at low concentrations. Treatment with parathyroid hormone did not stimulate adenylate cyclase activity. Thus, these cells are a well-differentiated, steroid-responsive clonal cell line that closely approximates the phenotype of the mature osteoblast. They should serve as an excellent model for the study of osteoblast biology.

AbstractAn analysis of the phospholipid, cholesterol, and phospholipid fatty acyl composition of isolated plasma membranes of 3T3 and SV3T3 mouse embryo cells has been performed. The results show that the plasma membrane of SV3T3 cells contain relatively less phosphatidylethanolamine and sphingomyelin and more cholesterol than 3T3 plasma membranes. The fatty acyl composition of individual phospholipid classes as determined by gas liquid chromatography also showed differences between 3T3 and SV3T3 plasma membranes. The plasma membranes of SV40 transformed 3T3 cells contain: (a) a higher percentage of 18∶1 and less 20∶3 and 20∶4 in phosphatidylethanolamine; (b) a higher percentage of 18∶1 in phosphatidylserine; and (c) a higher percentage of 18∶2 and 20∶4 in phosphatidylinositol.

AbstractThe lipid composition of plasma membranes isolated from differentialted and undifferentiated L6 myoblasts has been compared. In general, the plasma membranes of differentiated L6 myoblasts have a higher cholesterol to phospholipid molar ratio than plasma membranes of undifferentiated cells. Differentiated L6 myoblasts have increased relative amounts of phosphatidylethanolamine and phosphatidylcholine in their plasma membrane and a decreased relative amount of sphingomyelin when compared with the plasma membranes of undifferentiated myoblasts. In addition, preliminary results show that differentiated L6 myoblasts plasma membrane phospholipid shows differences in the fatty acyl composition, specifically there appears to be relatively more 17∶0 and 24∶1 and less 16∶1 and 18∶1 than in plasma membrane phospholipids of undifferentiated L6 myoblasts. These observations indicate that significant changes in plasma membrane lipid composition occur during myoblast differentiation. The role that changes in lipid composition play in control of cellular differentiation, however, remains to be elucidated.