Production of gamma interferon by human T and null cells and its regulation by macrophages

Human mononuclear subpopulations were tested for the capacity to produce interferon after mitogenic stimulation with protein A from Staphylococcus aureus. Mononuclear cells were separated into highly enriched macrophage, T-, B-, and null-cell subpopulations by Sephadex G-10 adherence, anti-human IgG F(ab′) two-column chromatography, and rosetting with sheep erythrocytes. Interferon (IFN) production was observed in both T- and null-cell preparations, but not in macrophage or B-cell preparations. Physicochemical and antigenic characterization of IFN from T- and null-cell preparations showed that both mononuclear subpopulations produced gamma IFN (IFNγ). Regulatory studies showed that IFNγ production was differentially regulated by macrophages. Macrophage addition to T lymphocytes augmented both cellular proliferation and IFNγ production, whereas macrophage addition to null cells suppressed IFNγ production and had no effect on the minimal proliferative response observed for these cells.     

Immunoglobulin properties of Epstein-Barr virus transformed human umbilical cord and adult peripheral blood lymphocytes

We have observed that a subset of E+ cells bind human monomeric IgG (FcR-IgG). In the present study, we have separated the E+, FcR-IgG+ cells by flow cytometry and tested their NK activity against the tumor KS62 using the chromium-release assay. Virtually all the NK activity residing within the E+ subset was mediated by E+, FcR-IgG+ cells. These findings are discussed in relation to the cellular lineage of the human NK cell.     

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.     

Differentiation of lens and pigment cells in cultures of neural retinal cells of early chick embryos

Dissociated cells of neural retinas of 3.5-day-old chick embryos (stages 20–21) were cultured as a monolayer in order to examine their differentiation in vitro. These cells started to grow actively soon after inoculation and formed a confluent sheet within which neuroblast-like cells with long cytoplasmic processes were differentiated by 8 days. At about 16 days the differentiation of both lentoid bodies and foci of pigment cells was observed, while neuronal structure disappeared. The numbers of lentoid bodies and foci of pigmented cells continued to increase up to 30 days, when primary cultures were terminated. The increase in δ-crystallin content, as measured by quantitative immunoelectrophoresis assay using rabbit antiserum against δ-crystallin, was consistent with the increase in the number of lentoid bodies in cultures. The amount of α-crystallin per culture, estimated by the same technique as above, reached a maximum at 16 days and decreased slightly during further culture. The differentiation of both lentoid bodies and pigment cells was observed also in cultures of the second generation. The results demonstrate that cells of the undifferentiated neuroepithelium of 3.5-day-old embryonic retinas can achieve at least three differentiations, neuronal, lens, and pigment cells, in vitro. We discuss several differences between the present results and the previous ones from in vitro cultures of 8- to 9-day-old embryonic neural retinas.     

Evidence for the identification of lymphocyte activating factor as the adherent cell-derived mediator responsible for enhanced antibody synthesis by nude mouse spleen cells

Human adherent peripheral blood leukocytes spontaneously elaborate both a thymocyte proliferative factor and a factor which augments the in vitro anti-sheep erythrocyte (SRC) plaque-forming cell (PFC) response of nu/nu mouse spleen cells. Nonadherent leukocytes do not spontaneously elaborate either factor. The adherent cell-derived factors appear to have an identical molecular weight (approximately 14,500 Daltons) as determined by Sephadex gel filtration. The data support the hypothesis that the molecule(s) mediating both enhancing activities is identical to the previously described adherent leukocyte product, LAF.     

Zinc accumulation and metabolism in primary cultures of adult rat liver cells. Regulation by glucocorticoids

Adult rat liver parenchymal cells were isolated by the collagenase perfusion technique and cultured as a monolayer for up to 20 h. The quantity of zinc accumulated from the extracellular environment was significantly increased by adding physiological concentrations of certain glucocorticoids to the medium. The degree of stimulation was directly related to glucocorticosteroids potency. Sex steroids, certain peptide hormones and prostaglandins E₂ and F_2α did not influence zinc accumulation.Control cells exhibited a decline of zinc accumulation after 4 h in culture although uptake processes were still operative. When dexamethasone, the most potent glucocorticoid used, was present in the medium the cells accumulated zinc at a linear rate greater than that seen in control cells, for at least 20 h. The dexamethasone-induced stimulation of zinc accumulation was relatively specific since ⁴⁵Ca, ¹⁴C-labelled amino acids and [³⁵S]cystine accumulation was not influenced by the hormone. A lag of 4 h was observed before an effect of dexamethasone on zinc accumulation could be detected. Moreover, the hormone-stimulated phase of accumulation was blocked when the cells were simultaneously incubated with either actinomycin D or cycloheximide. The additional complement of zinc accumulated by the dexamethasone-treated cells was localized in the cytosol fraction. Gel filtration and ion-exchange chromatorgraphy confirmed that this additional cytosol zinc was bound to metallothionein. [³⁵S]Cystine was incorporated into metallothionein in hormone-treated cells indicating that the protein was synthesized de novo during periods of enhanced zinc accumulation.     

An immunocytotoxicity assay for neural cells in monolayer cultures

The immunological analysis of cell surface constituents which may characterize neuronal and glial populations, though still in its infancy, will greatly facilitate the investigation of several important problems in neurobiology. One critical component of such analyses is the way by which a given antiserum can be shown to be active on, and possibly selective for neurons and glial cells from normal neural tissues. This report describes the use of monolayer cultures of normal neural cells for recognition and quantitative titration of antisera directed against them. Sera were collected from rabbits immunized with chick embryo spinal cord cell susptnsions, and found to be reactive to the same cells in the initial cell dissociate as well as in subsequent monolayer cultures of different in vitro ages. A monolayer assay procedure was developed, which (i) uses small numbers of cells and small volumes of immune reagents, with the possibility of further scaling down; (ii) applies equally to cultures using different substrata; (iii) permits differential counts of morphologically different cultured cells; (iv) allows to recognize cytological damage imposed by the immune serum in the presence, though not in the absence, of complement; and (v) quantitatively titrates the immune activity with 10- to 20-fold higher sensitivity than other titration procedures. While the study was not intended to investigate the possible specificities of the new antisera, it provided the unexpected observation that non-neuronal cells in these spinal cell cultures were considerably less sensitive than neurons to the complement-dependent action of the antisera.     

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.     

The use of hormone-supplemented serum-free media in primary cultures.

Recent advances in tissue culture and endocrinology have made possible the growth of established cell lines in hormone-supplemented serum-free media. The hormone requirements differ for different cell types but are similar or identical for the same cell types. The hormone supplements derived for four different cell types, a melanoma, GH3 pituitary tumor, and testicular cell lines TM3 and TM4 are used in preparing primary cultures for organs to detect melanoma metastasis, and grow normal pituitary and normal Leydig and Sertoli cells, respectively. This hormone supplementation and the concomitant elimination or reduction of the serum requirement is shown to have several advantages in the preparation of primary cultures including prolonged viability and function, partial or total selection of the desired cell type and inhibition of fibroblast overgrowth.It is felt that such culture systems will significantly expand the range of problems which can be approached using primary culture systems.     

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)’.     

Immunofluorescent and autoradiographic analysis of the relationship between the presence of histone H5 and DNA synthesis in developing chick erythroid cells

Simultaneous detection of histone H5 by indirect immunofluorescence and of [³H]thymidine incorporation by autoradiography on the same preparations of developing erythroid cells have been used to precisely define the extent of correlation between the loss of nuclear activity and the presence of histone H5. It was found that from day 3–12 of embryonic life there are two successive waves of double-labelled cells. At some stages, as many as 30% of the cells which incorporate [³H]thymidine also contain histone H5. Thus, the simple presence of H5 cannot be sufficient to cause nuclear inactivation. A kinetic analysis of the appearance and disappearance of [³H]thymidine-labelled cells, containing histone H5, and cells which are positive for both markers is presented. The result is consistent with the interpretation that the appearance of H5 in the first wave of double labelled cells occurs just before the erythroid cells become metabolically inactive. These observations modify the concept that histone H5 functions uniquely or solely as a template repressor.     

Autophagocytosis: in vitro induction by microtuble poisons.

In the rat liver in vivo autophagy has been shown to be induced by glucagon, cyclic AMP (cAMP), dibuturyl-cyclic AMP (db-cAMP) and vinblastine administration. Vinblastine and demecolcine were shown in this study to be potent inducers of autophagy also in Ehrlich ascites tumor cells in vitro. It is suggested that this effect is rather dependent on direct effect on membranes of endoplasmic reticulum than on the breakdown of microtubules by these compounds.     

The presence of the milk protein, alpha-lactalbumin and its mRNA in the rat epididymis

alpha-Lactalbumin, a modifier protein that changes the substrate specificity of galactosyltransferase, to promote the synthesis of lactose, is found in the mammary glands of lactating mammals and in milk. Molecules similar to mammary gland alpha-lactalbumin but distinct in their modifier activity have been found in rat epididymal fluid. We report here, using a rat mammary gland alpha-lactalbumin cDNA clone as a hybridization probe, RNA sequences homologous to alpha-lactalbumin mRNA were detected in total RNA from the rat epididymis. This finding suggests that alpha-lactalbumin or similar molecules, in addition to regulating lactose synthesis in the mammary gland, may have other important functions in mammalian reproduction.     

Colorimetric determination of catechol siderophores in microbial cultures

A highly sensitive spectrophotometric method for the selective detection of catechol compounds such as catechol siderophores (e.g., enterobactin) is described. The basis of the method involves the ability of the vicinal aromatic hydroxyl groups under acidic conditions to bring about a reduction of Fe3+ (from ferric ammonium citrate) to Fe2+. Detection of Fe2+ in the presence of Fe3+ is made with 1,10-phenanthroline under previously established conditions. The assay mixture is heated at 60 degrees C for 1 h to accelerate the development of color which is subsequently measured at 510 nm. The Beer-Lambert law is obeyed over the range of 0.16 to 60 microM 2,3-dihydroxybenzoic acid. Compared to the Arnow nitration method, the assay is more responsive, is approximately seven times more sensitive, and is effective with catechols substituted at positions 3 and 4. The method gives positive results with catechols such as DL-DOPA, L-dopamine, (+/-)-epinephrine, and DL-norepinephrine. Very rapid color development is obtained with ascorbic acid and p-diols, while m-diols are poorly detected. Low degrees of reactivity are shown by hydroxylamino and hydroxamate compounds. Phenolic, sulfydryl, indolyl, and quinonyl derivatives do not interfere with the reaction. The method has been adapted to determine catechol compounds in the culture medium of bacterial cells grown at different iron concentrations.     

Human lymphocyte subpopulations identified by bacterial adherence are functionally different.

Previously, two B (B₁ and B₂)- and four T (T₁, T₂, T₃, T₄)-lymphocyte subpopulations have been identified in human blood smears by bacterial adherence. Here, to study the functional differences between these subpopulations the T₁T₂ cells were separated from T₃T₄ cells by selective adherence to Escherichia coli-2⁴ monolayers. The adherent cells (T₁T₂ cells) responded well to concanavalin A in 3-day cultures and in mixed lymphocyte culture (MLC) in 6-day cultures and developed into cells specifically cytotoxic for allogeneic lymphocytes. The nonadherent cells (T₃T₄ cells) cultured for the same length of time were poorly responsive to concanavalin A, variably responsive in MLC, and poorly active in specific cytotoxicity. The T₃T₄ cells were naturally cytotoxic for allogeneic lymphocytes and for a normal lymphoblastoid cell line. We concluded that the T cells that bind E. coli-2 (T₁T₂ cells) are functionally different from those that do not bind (T₃T₄ cells).     

Selective emigration of suppressor T cells from Peyer's patches

The emigration of Peyer's patch lymphocytes to mesenteric lymph nodes was studied by injecting fluorescein isothiocyanate (FITC) directly into Peyer's patches. Using double immunofluorescence it was demonstrated that at 2 and 4 hr after FITC injection 70% of the labeled cells that migrated to mesenteric lymph nodes were T lymphocytes, although rat Peyer's patches contain only 15-20% T lymphocytes. At later time points after FITC injection this percentage of T cells derived from Peyer's patches gradually declined, most likely caused by selective interaction and/or retention inside the mesenteric lymph node. Determination of helper and suppressor T-cell subsets within this emigrating population showed an increased number of T suppressor cells migrating into mesenteric lymph nodes. The putative role of suppressor T cells in inducing systemic tolerance after oral antigen administration was discussed.     

Binding of nonsuppressible insulinlike activity to human serum. Evidence for a carrier protein.

When ¹²⁵I-labeled nonsuppressible insulinlike activity—soluble in acid/ethanol (NSILA-S) is incubated with human serum between 10 and 20% of the radioactivity are bound to serum proteins and can be displaced specifically by cold NSILA-S. Chromatography of the incubation mixture on Sephadex G-200 at pH 7.5 reveals three peaks of radioactivity in the large molecular weight region and a fourth one corresponding to low molecular unbound labeled NSILA-S. An excess of cold NSILA-S during preincubation leads to the disappearance of the two major large molecular weight peaks and to a concomitant increase of the peak eluting in the low molecular weight range. Binding of ¹²⁵I-labeled NSILA-S is highly sensitive to small concentrations of cold NSILA-S, whereas insulin, ACTH and human growth hormone are completely ineffective in displacing bound ¹²⁵I-labeled NSILA-S. NSILA-S preparations of different purity show displacement according to their specific biological activities. Furthermore, binding of ¹²⁵I-labeled NSILA-S to serum pH- and time-dependent and displays saturation characteristics. Chromatography of serum on Sephadex G-200 with 0.15 m acetic acid/0.15 m NaCl localizes the binding fraction in the 50,000–70,000 molecular weight range. Boiling of serum for 5 min abolishes binding completely.These studies help explain why the molecular weight of NSILA varied considerably from one group of investigators to the other.     

Characterization of ellipticine binding to native calf thymus DNA

The binding of [14C]ellipticine to native calf thymus DNA was studied using equilibrium dialysis. A Scatchard polt revealed the presence of high-and low-affinity binding sites in DNA, the former having a K of 4.0 X 10(7) M(-1) and an n (saturation limiting of binding) of 0.078 (1mol ellipticine/13 mol of DNA nucleotides). The forces involved in stabilizing the high-affinity binding, which has been equated with intercalative binding, were due to a combination of hydrophobic interactions and hydrogen bonding. Difference spectra of ellipticine in the presence of the polydeoxynucleotides, poly d(A-T) or poly d(G-C), showed that there was no base specificity involved in the high-affinity binding. Ellipticine binding to the low-affinity sites, which has been equated with surface binding, was due primarily to the participation of electrostatic interactions of ellipticine with the anionic phosphate groups on the double helical surface of DNA.