Human alveolar macrophages from bronchoalveolar lavage are usually poor accessory cells for antigen-induced T-lymphocyte proliferation and poor stimulators of allogeneic mixed leukocyte reactions (MLR) when compared to peripheral blood monocytes. In contrast, cells harvested from minced lungs are good stimulators of a MLR. We have characterized the accessory cells obtained after enzymatic digestion of human lung tissue. Pulmonary mononuclear cells were separated from the dissociated lung cell mixture on Ficoll-Hypaque. Loosely adherent cells (LAM) were obtained after an overnight incubation on plastic culture dishes of initially adherent mononuclear cells. LAM were significantly more effective than were pulmonary mononuclear cells (p less than 0.05), firmly adherent cells (p less than 0.05), alveolar macrophages obtained by bronchoalveolar lavage (p less than 0.05), or monocytes (p less than 0.05) in stimulating allogeneic resting T-cells. Addition of indomethacin and catalase markedly improved T-cell proliferation induced by LAM. Enrichment for Fc receptor negative or for nonphagocytic cells further enhanced the MLR-stimulating capacity of LAM. Phase-contrast studies demonstrated an enrichment in cells compatible with dendritic cells in LAM as compared to firmly adherent cells. We conclude that there are potent accessory cells in human lung that are loosely adherent, Fc receptor negative, and poorly phagocytic, and thus are dissimilar from classic macrophages. We hypothesize that cells similar to dendritic cells might play a role in the initiation of immune responses in lung parenchyma.
Growth of Mycoplasma pneumoniae was completely prevented by 0.06 gg of actinomycin D/ml, and 0.00375 ,g/ml caused 90% inhibition. It thus appears that M. pneumoniae is more susceptible to actinomycin D than previously reported. Low concentrations (0.019 ug/ml) of the antibiotic primarily inhibited ribonucleic acid synthesis and high concentrations (20 yg/ml) inhibited both ribonucleic and deoxyribonucleic acid synthesis.Mycoplasma pneumoniae appears to be more susceptible to actinomycin D than was previously reported by Tourtellotte (3). Because of Tourtellotte's report and the observation that actinomycin D (0.5 ,g/ml) inhibits glucose uptake of Escherichia coli spheroplasts (1), the present investigation was conducted.M. pneumoniae obtained from the American Type Culture Collection (ATCC 15293), was grown in Difco PPLO broth supplemented with 20% gamma globulin-free horse serum, 10% fresh yeast extract, and 0.5% glucose; the viable titer was measured in acid-forming units (AFU) with the aid of phenol red indicator.Actinomycin D (Merck Sharp and Dohme, West Point, Pa., and Calbiochem, La Jolla, Calif.) was dissolved in distilled water (100 ,ug/ml, stock solution), and the exact concentration of the stock solution was confirmed by optical density at 441 nm (e = 25.7 x 10').Direct exposure of antibiotic solutions to light was avoided at all times.The effect of actinomycin D on M. pneumoniae viability was determined by preparing dilutions of mycoplasma in growth medium containing the selected concentrations of the chromopeptide. All samples were incubated at 37 C and examined daily for acid production. Periodically, actively multiplying mycoplasma were checked for typical colonial morphology on a suitable agar-containing mycoplasma medium. AFU titers (log) were recorded on the 21st day of incubation.To facilitate the washing procedures utilized in the 3H-uridine or 3H-thymidine uptake experiments, M. pneumoniae was grown as monolayers in screw-cap tubes (16 by 125 mm) at 37 C. M. pneumoniae monolayers (10' to 109 376 AFU/tube) were exposed to various concentrations of actinomycin D. After 1 h of exposure to the antibiotic, 9H-uridine was added (final concentration, 2.5 ,uCi/ml) and incubation was continued. Samples were monitored for trichloroacetic acid-insoluble incorporation. Similar studies were conducted with 3H-thymidine, but a concentration of 4 ,uCi/ml was utilized. All samples were processed as follows. Triplicate tubes of each group were drained and washed with three 2-ml volumes of trichloroacetic acid. The trichloroacetic acid-insoluble fractions were drained and solubilized in 0.5 ml of hydroxide of Hyamine and 10 ml of scintillation counting fluid [2,5-diphenyloxazole, 4 g; 1,4-bis -(2-(4 -methyl-5-phenyl-oxazolyl)) -benzene, 200 mg; toluene, 950 ml; and absolute ethanol, 50 ml]. Activity per sample was monitored in a Packard Tri-Carb scintillation counter, model 3320.M. pneumoniae viability was completely inhibited by actinomycin D at a concentration of 0.06 sg/ml. Growth of this infectious agent wa...
The Smulow-Glickman gingival cell line (SmuLo0w and GLICKMAN, Proc Soc Exp Biol Med 121 :1294, 1966) would not be expected to function exactly like in situ gingival cells. However, this cell line is valuable as an in vitro model to study a number of dental problems pertaining to the gingiva. This cell line has provided a system for measuring cell adhesion to various substrata and it is also a suitable mediu1m for herpes simplex virus replication. These cells may be more like normal gingival cells than many wish to admit. A landmark characteristic of gingival epithelial cells is the desmosome which we demonstrate in the Smulow-Glickman cell line.This gingival cell line was grown in Earle's basal medium (BME) supplemented with 10% calf serum, 0.085%c NaHCQ1G and antibiotics (penicillin, 100 units/ml; streptomycin, 100 Mg/ml; and fLungizone, 25 pg/mI). Five milliliters of a suspension of cells (100,000/ml) were plated in 2 oz prescription bottles and incubated at 37 C until confluent monolayers were formed. Cells were prepared for electron microscopy by briefly treating the monolayers with a dilute solution of trypsin (0.025% ) to release small groups and sheets of cells from the growth substrate. While this mild enzymatic treatment of cells does effect cell surface topography, internal ultrastructural details are not noticeably affected. Suspended cells were fixed in 2% glutaraldehyde, followed by 1 % osmium tetroxide (both fixative solutions were made up in Hanks' balanced salt solution). The cells were then rinsed and dehydrated through a graded series of ethanol to propylene oxide. After infiltration with Epon, the cells were forced into the bottom of embedding capsules by centrifugation and placed in an oven to polymerize. Thin sections were stained with lead citrate and uranyl acetate. Cells were observed and photographed using an electron microscope.With the light microscope, the cultured cells were observed to form a continuous sheet of polygonal cells, typical of epithelial cells in culture. Ultrastructurally, the cells were found to contain an abundance of mitochondria, microtubules, and microfilaments. Many cells contained autophagocytic vacuoles in which there were lamellar inclusion bodies and vestiges of cellular organelles. A well-developed Golgi apparatus was often seen. The endoplasmic re-1106 ticulurn was relatively Lundeveloped. Smooth endoplasmic reticulum was scen ustually in association with the Golgi apparatus. Profiles of rough endoplasmic reticuluin were rarely inoted. Monoand polyribosomes were observed throughout the cytoplasm. Nuclear chromatin was predominantly granular with minimiial clumping at the nuclear membrane. Nucleoli were unremarkable. The most outstanding ultrastructuLral feature of the cultLured cells was their attachment to one another by means of desmosomes. These
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