Absence Of Other Cell Types Research Articles

Expression of tracheal differentiated functions in serum-free hormone-supplemented medium.

Most dissociated airway epithelial cells in culture express few of their in vivo functions and only to a limited degree. In this report, we demonstrate that hamster tracheal epithelial (HTE) cells cultured on a collagen gel substratum in a serum-free hormone-supplemented medium differentiate to cilia-beating and mucus-secreting cell types. The medium is Ham's F-12 supplemented with insulin, epidermal growth factor, transferrin, hydrocortisone, cholera toxin, bovine hypothalamus extract, and vitamin A. Under these culture conditions, HTE cells exhibit a growth rate of 24 h/population doubling and reach confluency, at a density of 2-5 X 10(4) cells/cm2, within 2 weeks. Both the collagen gel substratum and vitamin A of this culture system are important to the growth and differentiation of HTE cells in vitro. Evidence of HTE cell differentiation has been obtained at both the ultrastructural and the histochemical levels. In addition, a variety of biochemical studies (gel filtration, ion exchange column chromatography, enzyme digestion, nitrous acid treatment, and composition analysis) indicate the production of mucin-like glycoprotein in the HTE cultures. The levels of mucin-like glycoprotein were found to closely correlate with the histochemically quantitated levels of the mucous cell type. Kinetic studies demonstrate that HTE cells rapidly lose their differentiated features during the attachment stage of primary culture but redifferentiation occurs after the cultures reach confluency. The ability of HTE cells to grow and differentiate in this serum-free culture system in the absence of other cell types should greatly facilitate the study of mucociliary functions in vitro.

Direct stimulation of adcc by cloned gamma interferon is not ablated by glucocorticoids: Studies using a human monocyte-like cell line (U-937)

We have used the macrophage-like cell line U-937 to demonstrate that recombinant gamma (immune) interferon (γ-IFN) acts directly on the mononuclear phagocyte in the absence of other cell types to increase Fc receptor sites and antibody-dependent cellular cytotoxicity (ADCC). Incubation of U-937 for 18hr with 2% γ-IFN-rich supernatant, or with 10 U/ml of pure recombinant γ-IFN, resulted in a seven-fold increase in Fc receptors as measured by the binding of radiolabeled IgG or fluoresceinated IgG and cytofluorography. Simultaneous measurement of ADCC for chick erythrocytes showed a seven-fold increase. This augmentation of Fc receptors and function was not ablated by an immunosuppressive concn of the glucocorticoid dexamethasone. The potent effects of γ-IFN both on surface receptors and effector functions of macrophages suggest that it is an important mediator in the efferent limb of immunity. Moreover, our findings that physiologic levels of glucocorticoids do not block activation of the mononuclear phagocyte support our view that glucocorticoids are immunosuppressive as a result of their action on γ-IFN-producing cells. This would seem an important consideration in the development of potential strategies for obviating steroid-induced immunosuppression.

Disparate functional properties of two interleukin 1-responsive Ly-1+2- T cell clones: distinction of T cell growth factor and T cell-replacing factor activities.

We describe the properties of two Ly-1+2- T cell clones (Ly-1.14 and Ly-1.21), which are maintained in long-term culture in the absence of other cell types. The clones require media containing a source of interleukin 1 as well as interleukin 2. They retain physiologic responses to interleukin 1, which is required for optimal production of T cell lymphokines by these clones in response to concanavalin A (Con A). The two Ly-1+2- T cell clones differ in their production of lymphokines after stimulation by Con A. The supernatant of clone Ly-1.21 promotes the proliferation of T cells maintained in long-term culture, induces antibody synthesis in cultures of B cells and antigen, and induces the differentiation of cytolytic cells in cultures of thymocytes and antigen; these assays define the properties of T cell growth factor (TCGF), T cell-replacing factor for B cells (TRF-B), and T cell-replacing factor for cytolytic cells (TRF-C), respectively. In contrast, the supernatant of clone Ly-1.14 contains only TCGF activity and does not promote antibody synthesis by B cells or differentiation of cytolytic cells from thymocytes. The results indicates that TCGF and TRF activities reside on independent, although perhaps related, molecules.

Suspension culture of separated follicles consisting of differentiated thyroid epithelial cells.

We have prepared thyroid follicles in suspension culture to use as a model system in vitro for investigation of some properties of the thyroid gland. The follicles were free of endothelial cells, fibroblasts, and other nonepithelial cells. They were prepared by collagenase treatment of minced rat thyroid glands followed by differential filtration of the suspension through nylon meshes. Small clusters of principal thyroid epithelial cells were separated from large fragments and single cells. They were cultured in dilute suspension in Coon's modified F-12 medium in dishes coated with agarose to avoid having the cells attach to the dishes. By culture day 3, most of the clusters formed closed follicles containing a periodic acid-Schiff-positive colloid but without a basal lamina. Follicle walls contained an occasional C cell. The epithelium resembled that in the thyroid of a recently hypophysectomized rat, with normal polarity and organelle complement normal with respect to position and abundance, with basally located lysosomes, no pseudopods, and no colloid droplets. The cells were responsive to thyroid-stimulating hormone (thyrotropin) and to dibutyryl cyclic AMP. Thyroid-stimulating hormone at 10 munits/ml resulted in apical migration of lysosomes and formation of pseudopods and colloid droplets within 30 min; longer exposure resulted in depletion of luminal colloid. The results indicate that the suspended follicles resemble follicles in vivo with respect to morphology and responsiveness to thyroid-stimulating hormone in the absence of other cell types.

Environmental Determination of Neurotransmitter Functions in Developing Sympathetic Neurons

One area of neuronal development which is particularly interesting is the interaction between cells and their environment as they migrate and differentiate. One of the techniques that has proven useful in studies of such interactions is cell culture, because of the opportunity to control both the fluid and cellular environments surrounding developing neurons. It is the ability to grow dissociated neurons in culture, either in the virtual absence of other cell types, or in the presence of any of a variety of known types of non-neuronal cells that enables to control the type of transmitter produced and the type of synapse formed by developing sympathetic neurons in culture. When sympathetic neurons are cocultured with appropriate non-neuronal cells, they develop striking cholinergic properties: they synthesize and store large amounts of acetylcholine (ACh) and use it at functional cholinergic synapses with a number of targets. Furthermore, this cholinergic induction caused by non-neuronal cells can be mediated by a diffusible factor. Neurons grown in culture medium that has been conditioned by incubation on cultures of appropriate non-neuronal cells (CM) also causes dramatic increases in (i) choline acetyltransferase activity (CAT) as measured in neuronal extracts, (ii) ACh synthesis and accumulation from [ 3 H] choline by living cells, and (iii) cholinergic synapse formation between the neurons. Thus the presence of certain non-neuronal cells, or a medium conditioned by them, has a profound effect on the type of transmitter chosen by the sympathetic neurons.

Survival and development of ciliary ganglion neurones grown alone in cell culture.

NEURONAL cell death is a conspicuous part of development for many neuronal populations in the vertebrate nervous system1,2. Although little is known about the mechanisms that control neurone death, it seems that interactions with the postsynaptic target tissue are important. Thus, for the chick ciliary ganglion (CG), Landmesser and Pilar showed that half of the neurones present die between days 8 and 13 in the embryo. Before cell death, all the neurones extend processes to the periphery. Ablating the periphery early in development causes more than 90% of the neurones to die at the normal time3. We have previously shown that nearly all the neurones from chick CG, including those destined to die in vivo, can survive and develop in cell culture when ganglia from 8-d-old embryos are dissociated and grown with skeletal myotubes4. Many of the neurones innervate myotubes in these conditions. Are neuromuscular synapses necessary to prevent the death of ciliary ganglion neurones? We report here that they are not. Culture medium conditioned by contact with heart or skeletal muscle cells permits complete long-term survival of the dissociated neurones grown in the absence of other cell types. The neurones develop levels of choline acetyltransferase (CAT) activity comparable to those obtained for neurones grown with myotubes.

Studies on rat sympathetic neurons developing in cell culture: I. Growth characteristics and electrophysiological properties

In this series of three papers, we describe electrophysiological and pharmacological studies on sympathetic principal neurons developing in cell culture. This paper is concerned with the methods for growing and recording from the neurons and with observations on some of their electrical properties. The succeeding papers are concerned with functional synapses which the neurons form with one another. Superior cervical ganglia of newborn rats were dissociated into single cells and small cell clusters, and the resulting cell suspension of principal neurons and a much smaller number of non-neuronal cells was cultured at low density in medium containing nerve growth factor ( D. Bray, 1970, Proc. Nat. Acad. Sci. USA. 65, 905–910; R. E. Mains and P. H. Patterson, 1973a, J. Cell Biol. 59, 329–345 ). As in the previous studies the multiplication of the non-neuronal cells could be controlled so that the neurons grew in the presence of an increasing number of non-neuronal cells or in the virtual absence of other cell types. Another method for obtaining mixed cultures was to plate the initial cell suspension onto a preexisting layer of cells dissociated from some other tissue (e.g., heart). Neurons grown for 3 weeks or longer in the presence of non-neuronal cells had resting potentials, passive electrical properties, and action potentials generally similar to those reported for principal neurons of the superior cervical ganglia of adult rats. Through the use of tetrodotoxin, tetraethylammonium, and cobalt, evidence was obtained for the presence of potential-sensitive sodium, potassium, and calcium channels. Frequently the action potential was followed by a prolonged after-hyperpolarization whose properties suggested the presence of potassium channels controlled by calcium ions. When the neurons were grown in the absence of non-neuronal cells, the action potentials were similar, but the prolonged after-hyperpolarization was rarely seen, and the neurons usually discharged repetitively in response to a steady depolarization.

The induction of acetylcholine synthesis in primary cultures of dissociated rat sympathetic neurons: II. Developmental aspects

Dissociated sympathetic neurons from the neonatal rat, grown in cell culture in the virtual absence of other cell types, can develop many of the properties expected of differentiated adrenergic neurons including the ability to synthesize and accumulate catecholamines (CA) 2 2 Abbreviations used: Acetylcholine, ACh; conditioned medium, CM; catecholamine, CA; choline acetyltransferase, CAT. . However, in the presence of high concentrations of appropriately conditioned medium (CM), the cultures develop the ability to synthesize and accumulate acetylcholine (ACh); correspondingly, their ability to synthesize CA decreases. In this paper several developmental aspects of the CM effect are described. The time course of development of cultures grown with or without CM was followed using synthesis and accumulation of [ 3H]CA from [ 3H]tyrosine and production of [ 3H]ACh from [ 3H]choline as assays for adrenergic and cholinergic differentiation. The ability to produce CA or ACh developed along parallel time courses in the two sets of cultures, rising primarily during the second week in vitro and reaching a plateau during the fourth week. When CM was used as a cholinergic developmental signal, the sympathetic neurons showed a decreasing response to addition of CM as they matured adrenergically; addition of CM during the third or fourth 10 days in vitro was not as effective in inducing ACh production as addition during the first or second 10 days. Similarly, removal of CM at various times from cultures previously grown in CM showed that the cholinergic induction caused by CM was not easily reversible in older cultures. Thus, as with the adrenergic decision, the cholinergic decision becomes less reversible as the phenotype becomes fully expressed.

Antibody-Dependent Killing of Erythrocyte and Tumor Targets by Macrophage-Related Cell Lines: Enhancement by PPD and LPS

Five murine macrophage or monocyte-related tumor cell lines in culture were compared for antibody-dependent lysis (ADL) of a tumor target (T lymphoma EL4) and lysis and phagocytosis of sheep erythrocytes (RBC). Some lines were effective only with RBC targets, while others were capable of lysing these and tumor targets. Both murine alloantisera to H-2 and Thy 1.2 antigens on the target cells and rabbit anti-mouse spleen and anti-mouse thymus sera directed target lysis, while normal mouse or rabbit sera were inactive. Preincubation of macrophage line RAW264 with lipopolysaccharide (LPS) or purified protein derivative from Mycobacteria (PPD) for 2 days resulted in an average of 76% or 86% increase, respectively, in ADL of RBC, although there was no stimulation in RBC phagocytosis or in nonspecific or antibody-dependent lysis of tumor targets. These results indicate that macrophage cell types are capable of antibody-dependent tumor lysis, that macrophages are probably heterogeneous in effector cell activities, and that they can be stimulated to increased ADL in the complete absence of other cell types.