Concanavalin A mediated attachment and ingestion of red blood cells by macrophages.

The interaction of red blood cells and macrophages mediated by Concanavalin A (ConA) was studied using mouse peritoneal macrophages and fresh, homologous red cells. Erythrocytes exposed to ConA at 0.5 μg/ml, a condition that leads to a saturation of 3% of the ConA sites, were bound by macrophages at 22 °C. The ConA inhibitor, α-methylmannoside, prevented this attachment of red cells and largely reversed it when added to preformed macrophage-red cell rosettes up to 90 min. However, red cell attachment was essentially irreversible by 3 h. Electron microscopy showed a progressive increase in the degree of contiguity between red cells and macrophages with time, some macrophage projections distorting and partially encircling red cells at 3 h. Macrophages pretreated with high concentrations of ConA (25 μg/ml) also bound red cells. However, phagocytosis of adherent red cells did not occur at either 22 or 37 °C, even when both red cells and macrophages were pretreated with ConA. In contrast, phagocytosis of attached red cells was observed when preformed rosettes were exposed to ConA at a concentration of 5 μg/ml, and it was complete with ConA at a concentration of 25 μg/ml. These studies demonstrate that ConA in low concentration on red cells is detected by macrophages which form a progressively tighter bond with the red cell surface. However, it appears that phagocytosis can occur only under conditions in which a high density of ConA is established on the surface of the red cell.     

Bacteriophage T4 tail length is controlled by its baseplate

Purified T4 baseplate, when treated with high concentrations of pancreatic RNase, are inactive in $\text{in}\text{vitro}$ complementation assays. Their ability to initiate tail tube assembly is not altered; but the most probably length of the tube-baseplate formed is only 800A, compared to 1000A, the normal tube length, when untreated baseplates are used. Thus, baseplates help to regulate tube length, possibly by a template mechanism. Several minor baseplate proteins which may be involved in determining the length, including gp54, are missing from RNase - treated base-plates. These effects may be due to an unidentified protease contaminant of the RNase, since they are inhibited by phenylmethane sulfonyl fluoride.     

The induction of suppressor T cells by lipopolysaccharide in human peripheral blood lymphocyte cultures in the presence of fetal calf serum

Alveolar macrophages (AM) were collected by repeated endobronchial lavage from mice, rats, guinea pigs, and rabbits, and titrated into cultures of mitogen-stimulated syngeneic or autochthonous lymphocytes. Significant species differences were detected in regard to AM activity in the cultures. AM from guinea pigs and mice stimulated PHA-induced lymphoproliferation, while those from rats and rabbits were inhibitory; blood or peritoneal macrophages were not inhibitory in any of the species examined.     

The uptake of cyclic AMP by human erythrocytes and its effect on membrane phosphorylation.

The effects of time and cyclic AMP concentration on cyclic AMP uptake and membrane phosphorylation were studied using intact human erythrocytes. The rate of uptake of cyclic [³H]AMP was nearly linear with respect to cyclic AMP concentration. The amount taken up was small compared to the extracellular cyclic AMP concentration, but was sufficient to significantly increase the intracellular cyclic AMP concentration. Incubation with cyclic AMP resulted in increased incorporation of ³²P_i into several phosphorylated membrane peptides of the intact erythrocytes. Although cyclic AMP altered the distribution of radioactivity among the membrane components, the total amount of incorporation was not increased. The effect of cyclic AMP on phosphorylation of membrane peptides was observed with extracellular cyclic AMP concentrations as low as 1 μm and was most pronounced in incubations of 1 to 4 h. These results indicate that cyclic AMP can enter erythrocytes in sufficient amounts to alter the activity of cyclic AMP-dependent protein kinases, or to alter the rate of turnover of certain phosphorylated membrane peptides.     

Production of human/mouse hybridomas secreting a human lymphokine,osteoclast activating factor

A human/mouse hybridoma was developed which has the property of secreting a human bone resorbing factor similar or identical to the osteoclast activating factor (OAF) isolated from human tonsil lymphocytes. Mouse plasmacytoma cells negative for OAF production were fused with an enriched subpopulation of human tonsil lymphocytes that had been activated with phytohemagglutinin (PHA) to produce OAF (G. E. Nedwin, M. A. Mohler, and R. A. Luben, submitted for publication). Culture supernatants from mixed hybridomas contained a bone resorbing protein shown to cause the release of ⁴⁵Ca from previously labeled mouse calvaria. The bone resorbing activity from these hybridomas was inhibited by the presence of OAF-specific monoclonal antibodies. Several hybridomas retained OAF production following limited dilution cloning. One clone, CD6.20, showed a biphasic dose-response curve for bone resorption similar to that of purified OAF from PHA-activated human tonsil lymphocytes. OAF production in the CD6.20 cell line has been retained for over 100 passages. Karyotype analysis of this cell shows the presence of human chromosomes 10 and 18 and the X chromosome.     

Increase in DNA synthesis in Werner''s syndrome cells by hybridization with normal human diploid and HeLa cells

The dominance or recessiveness of the senescent phenotype in cells from patients with Werner's syndrome (WS cells) was investigated using cell fusion. The [³H]thymidine labeling index of normal human diploid fibroblast cell X WS cell heterodikaryons was considerably lower than that of normal homodikaryons, but was significantly higher than that of WS homodikaryons. The labeling index of WS cell X HeLa cell heterodikaryons was the same as that of HeLa homodikaryons. The labeling indices of heterodikaryons obtained by fusion between various strains of premature aging cells were as low as those of parental homodikaryons. These results indicate: (1) the senescent phenotype of WS cells appears to be partially recessive to the phenotype of normal cells and completely recessive to that of HeLa cells; (2) the marked inhibition of DNA synthesis in normal nuclei in heterodikaryons with WS cells could be due to ‘senescent factor(s)’ in WS cells; and (3) no complementation phenomenon was observed among genetically different premature aging cells, probably due to ‘senescent factor(s)’.     

Ia-positive T lymphocytes are the producer cells of interferon gamma

The production of γ-interferon (IFNγ) in human peripheral blood T lymphocytes was induced by stimulation with PHA. For identification of the producer cell of IFNγ, double fluorescence studies were undertaken and titers of interferon were determined in preparatively separated T-cell subpopulations reactive with one of the monoclonal antibodies OKT3, OKT4, OKT8, and OKIa1. Production of IFNγ was found in OKT3⁺, OKT4⁺, and OKT8⁺ cells. However, IFNγ production occurred only in T cells also reactive with the monoclonal antibody OKIa1. Addition of macrophages had no substantial effect on interferon titers in these subpopulations. It is suggested that the T cell subset producing IFNγ is characterized by its reactivity with the monoclonal antibodies OKT3, OKT4 or OKT8, and OKIa1.     

Changes in ultrastructures and enzyme activities during differentiation of myeloid leukemia cells to normal macrophages

Changes in ultrastructures and in enzyme activities were investigated electron microscopically, cytochemically and biochemically when mouse myeloid leukemia cells, Ml cell line, successfully differentiated to normal macrophages after incubation with a conditioned medium harvested from secondary embryo fibroblasts, or a lipopolysaccharide from Salmonella typhosa. The number of mitochondria increased significantly accompanied by the enhanced activity of cytochrome oxidase per cell, although the activity in each mitochondrion remained unchanged. The rough-surfaced endoplasmic reticulum elongated and often exhibited a concentrically multilayered lamellae. Glucose-6-phosphatase activity, a marker enzyme for the endoplasmic reticulum, also increased. Primary lysosomes were newly formed where acid phosphatase activity was positively demonstrated. Ten-nm cytoplasmic microfilaments, mainly forming bundles, and other microfilaments less than 6 nm wide were formed newly and abundant. Budding of type C viruses from the plasma membranes was reduced strikingly. Another established cell line, Mm-1, which spontaneously differentiated from the Ml cell line, was characterized completely by a macrophage, in which azurophilic granules (primary lysosomes), secondary lysosomes possessing strong activity of acid phosphatase and 10-nm microfilaments were most remarkable. These non-transplantable Mm-1 cells sometimes exhibited budding of viruses.     

Arrested states produced by isoleucine deprivation and their relationship to the low serum produced arrested state in Swiss 3T3 cells

This study examines cell cycle maturational arrests induced by isoleucine deficiency in Swiss 3T3 cells. Whereas low serum selectively blocks the maturation of cells in mid-G1, while allowing late G1, S, G2 and M cells to continue maturing through the cell cycle, isoleucine deprivation blocks the maturation of cells at the end of G1 and also in late S. These blocks are at median ages of approx. 5.2 ± 1.3 h and 12.1 ± 3 h after division. Cells prevented from maturing beyond these two points require serum to resume proliferation, although they have passed the mid-G1 low serum block point. This indicates that resumption of proliferation requires additional events not part of normal transit through the cell cycle. Furthermore, the kinetics of growth resumption differ from those of low serum arrested cells. Cells arrested by isoleucine deprivation appear to be in physiological states which are not part of the proliferative cell cycle and which do not coincide with the low serum arrested state.     

Use of intact rat brain cells as a model to study regulation of muscarinic acetylcholine receptors

Intact rat brain cells were dissociated and used to study the regulation of muscarinic acetylcholine receptors upon exposure to muscarinic receptor agonists. Incubation of cells with carbamylcholine resulted in a time-dependent decrease in subsequent [3H]N-methylscopolamine specific binding, an effect which reached a steady state after 3 hr at 37 degrees C. This effect of carbamylcholine was dependent on the concentration of the agonist in the incubation medium and was due to a reduction in the maximal binding capacity of the receptor with no decrease in the affinity of the remaining receptors. This preparation might be useful in future studies to elucidate the mechanisms underlying the regulation of muscarinic acetylcholine receptors in the central nervous system.     

Selective recognition in erythrophagocytosis by mouse peritoneal macrophages. Isolation and purification of a possible self-marker from mouse erythrocyte membranes

The percental participation of exogenous cytidine in liver RNA synthesis was determined after application of ³H-cytidine to rats. The amount of exogenous cytidine was varied by a factor of 5 × 10⁵, between 0.000 02 and 10.0 μg/g rat. With the ³H-cytidine doses and specific activities most frequently reported in the literature, the percental participation of the exogenous precursor is only about 0.1%, with 99.9% of the cytidylic acid incorporated into RNA under these conditions being of endogenous origin.The results show that the upper limit of the tracer dose of exogenous cytidine is about 1.0 μg/g rat. Within this tracer region 1.8% of ³H-activity—and therefore 1.8% of the amount of exogenous cytidine—is incorporated into liver RNA. The dependence of the percental participation on the duration of the experiments is examined.It is shown that autoradiographic grain density and specific activity of RNA can only be regarded as direct measures for the rate of RNA synthesis in different cells and animals if the percental participation of exogenous cytidine in RNA synthesis is generally of equal value.Comparable situations exist in the incorporation of ³H-thymidine into DNA as shown by earlier experimental work.     

Production and perception of pheromones by the beetle Tribolium confusum

Adults of Tribolium confusum secrete two pheromones. The first, produced by the male, is attractive to both sexes and the second, produced by the female, is attractive to the male only. Pheromone production and perception was studied in relation to habituation, beetle age, time of day and previous mating. A living source of each pheromone habituates the responding beetles, the male pheromone habituating more strongly; female pheromone habituates only in the absence of the male pheromone. Habituation to one pheromone was always accompanied by an enhanced response to the other.Five days after emergence, production of male pheromone reaches a peak that is maintained. Production of female pheromone peaks after 3 days. Both sexes are responsive to male pheromone immediately upon eclosion, males reaching maximum response at 14 days, females at 8 days. Males are also responsive to female pheromone upon eclosion reaching maximum response at 8 days; female response to female pheromone is imperceptible. Males but not females display a 24 hr rhythm in pheromone production. Mated beetles did not differ significantly from unmated beetles in their ability to perceive pheromones. Alteration in male pheromone production after mating was detected by females but not males; this pheromone may, therefore, act as both a sex and aggregation pheromone.     

Antiproliferative effect of interferon on a Burkitt''s lymphoma cell line

The effect of interferon (IF) on the growth of a Burkitt's lymphoma cell line was analysed. The degree of depression of cell doublings was the same if the cells were in a steady state mode of exponential growth or in a resting state (G0) when IF was added. As IF had a lag time of 24 h before decreased growth could be observed, cells in G0 did not seem to be more sensitive when growth was estimated by cell counts expressed as cell doublings. IF inhibited cells to proceed into the cell cycle and the possibility that IF may increase the escape into a G0 loop is discussed.     

Time-dependent loss of prostaglandin E release by adherent cells in response to stimulation by thyroid cells: Prevention of this loss by phytohemagglutinin

Preplating human adherent peripheral blood mononuclear cells (PBMC) for up to 24 hr results in a progressive decrease in their basal PGE release, and in the loss of their ability to increase PGE release during a subsequent 72-hr coculture period with allogeneic human thyroid cells. Phytohemagglutinin (PHA) present during a 24-hr adherent-cell preplating period prevents, in part, the loss of this PGE response to thyroid cells. These data indicate that adherent cells require continual stimulation by the thyroid cells or by PHA in order to maintain their ability to increase PGE secretion in response to thyroid cells.     

C3-Activating proteases on human lymphoblastoid cells superinfected with epstein-barr virus

The role played by macrophages in feedback inhibition of the immune response during murine brucellosis, allowing establishment of chronic infection, was investigated using a number of approaches. First, it was shown that the degree of splenomegaly (a measure of macrophage influx) following infection with Brucella abortus strain 19 did not correlate with the course of bacterial numbers in the spleen of CBA, BALB/c, and C57B1/10 mice. Second, it was shown that a rough, mucoid mutant, B. abortus strain 19R, although causing a chronic infection, did not induce splenomegaly. Nor could “suppressor macrophages” be demonstrated in these spleens. Delayed-type hypersensitivity during this infection was lower than with B. abortus strain 19. Third, no nonspecific suppression of unrelated immune responses in CBA mice infected with B. abortus strain 19 could be demonstrated, despite the very large numbers of macrophages in the spleen. The responses tested included delayed-type hypersensitivity and cell-mediated resistance to Listeria monocytogenes, antibody response to sheep erythrocytes (both serum antibody and plaque-forming cells in the spleen) and skin-graft rejection.     

The development of granulomatous pulmonary inflammation in rabbits by aerosol challenge: I. Release of plasminogen activator by alveolar macrophages

A rabbit model of hypersensitivity pneumonitis (HP) was employed to evaluate the release of plasminogen activator (PA) as a method for monitoring the degree of pulmonary inflammation. PA release from alveolar macrophages (AM) was shown to coincide with inflammation and was maximal at approximately 2 weeks of aerosol challenge. PA release could also be induced in normal AM by peripheral lymphocytes obtained from sensitized animals after incubation with antigen. Unseparated peripheral blood mononuclear cells from experimental animals also exhibited antigen-induced PA release. These results suggest that the measurement of PA release using several different cell populations can be used to evaluate pulmonary inflammation in HP.     

Alveolar macrophage lipoxygenase products of arachidonic acid: isolation and recognition as the predominant constituents of the neutrophil chemotactic activity elaborated by alveolar macrophages

Human immune interferon preparations have anticellular activity on human cell lines (WISH and HEp-2). This anticellular activity copurified with the human immune interferon and appears to be a function of the immune interferon molecule. On the basis of a unit of antiviral activity, purified human immune interferon had about 20 and 100 times more anticellular activity than purified fibroblast or leukocyte interferon, respectively. The possible implications of this finding in the treatment of human neoplasia are discussed.     

Postfreeze viability of human renal epithelial carcinoma cells related to phase transformations in ternary solutions

The postfreeze viability of human renal epithelial carcinoma cells frozen in solutions based on a complex physiologic support medium to which additions of NaCl and a cryoprotective agent, either glycerol or dimethyl sulfoxide (DMSO) were made, have been determined by a dye exclusion technique. The support medium consisted of either Eagle's Minimum Essential Medium with Hanks' salts added (MEM) or this same medium supplemented with 20 vol% heat-inactivated fetal calf serum (MEM + FCS). Glycerol was found to be an ineffective cryoprotective agent for these cells, while DMSO was highly effective. Addition of NaCl along with the DMSO further improved the viability of cells frozen at −196 °C. Freezing and thawing rates were found to be important with a slow freezing rate, 2.5 °C/min, and a rapid thawing rate, 240°C/min, yielding the best results.Maximum viability occurred in solutions containing 80 to 95 wt.% (MEM + FCS) with the balance being DMSO and NaCl in the weight ratio of 9:1. In addition to primary ice formation, two nonequilibrium glassy phases were observed during DTA studies of these solutions (10). The exintence of these vitreous states reduces the chances thet cells will be exposed to hypertonic concentrations of salt in the extracellur fluids during freezing-out of primary ice.