Mitogen-stimulated Human Lymphocytes Research Articles

T cell growth factor-enhanced PHA response of human thymus cells: requirement for T3+ cells.

Human thymocytes normally proliferate poorly in PHA-stimulated cultures, as measured by thymidine uptake. Their response increases when supernatant factors from mitogen-stimulated human lymphocyte cultures (SUP) are added. The cells that proliferate in PHA + SUP-stimulated cultures continue to divide if they are resuspended with fresh SUP every 3 to 4 days. PHA-stimulated thymocytes are deficient in the production of the T cell growth factor interleukin 2 (IL 2). The phenotype of thymocytes prior to stimulation was 90% T6+, 80% T4+, 83% T8+, and 12% T3+, whereas that of the cells after 3 days in culture with PHA + SUP was 1% T6+, 18% T4+, 66% T8+, and 80% T3+. Lysis of T3+ cells from the thymocyte preparation before culture greatly reduced the proliferative response to PHA + SUP. This suggests that the PHA-induced proliferation of more mature thymus cells is restricted by a lack of essential growth factors (either IL 1 or IL 2) and that the co-stimulating activity of SUP on human cells is predominantly on thymus cells that already are T3+.

Determination of IgG subclasses and Gm allotypes in culture supernatants of pokeweed mitogen-stimulated human blood lymphocytes.

Concentrations of the 4 IgG subclasses, of IgM, and of IgA were determined in culture supernatants of pokeweed mitogen-stimulated mononuclear cells isolated from human peripheral blood. Time-course studies showed cumulative secretion of IgM, IgA, and the subclasses IgG1, IgG2, and IgG3 but not of IgG4. In supernatants of 7-day cultures of unseparated mononuclear cells, IgG1 was predominant, followed by IgG2 and IgG3. Removal of most monocytes from the mononuclear cells resulted in an overall decrease of immunoglobulin production but had no influence on the relative IgG subclass distribution. In supernatants of T cell-enriched cultures, the synthesized IgG was deficient in IgG1. Likewise, in supernatants of B cell-enriched cultures, IgG2 exceeded IgG1 concentration. Elimination of the T gamma cells suggested some influence of this subset on IgG2 production. Gm allotypes determined in supernatants corresponded to those in serum of the blood donors.

Inhibition of lymphocyte proliferation by histamine and related compounds not mediated via H1- or H2- receptors.

1 The effects of histamine and chemical analogues were examined on mitogen-stimulated human lymphocyte proliferation. 2 Compounds with selective agonist activity at either H1- or H2-receptors were found to inhibit proliferative responses, although N-3-methyl-histamine which does not act on either receptor was as inhibitory as histamine itself. 3 The H2-receptor agonist, dimaprit, had a profound inhibitory effect on proliferation, however nordimaprit, which has little or no H2-agonist activitiy, was more active on lymphocytes. Impromidine, although a potent H2-agonist, failed to produce such inhibition. 4 The effects of dimaprit and nordimaprit were not reversed by H2-receptor antagonists, cimetidine or metiamide. 5 These results do not support the view that the antiproliferative effects of histamine and related compounds are mediated via conventional H1-or H2-receptors. 6 SKF 93390 was found to be the most active of the dimaprit analogues tested, which could represent a novel series of potential immunosuppressive agents.

Membrane protein synthesis in mitogen-stimulated human T lymphocytes.

Proteins were biosynthetically labeled with [35S]-methionine in resting and mitogen-stimulated human peripheral blood lymphocytes. Lymphocytes were disrupted by sonication, the membrane fraction was isolated by differential centrifugation, and radiolabeled membrane proteins were identified after 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and autoradiography. The T cell mitogen concanavalin A (Con A) produced selective increases in the incorporation of [35S]-methionine into a membrane protein (or family of 4 structurally related membrane proteins) with an apparent m.w. of 28,000 and approximate isoelectric points (pI) of 5.5, 5.7, 6.0, and 6.2 (p28). Quantitative microdensitometry was used to evaluate p28 synthesis in a large number of different lymphocyte donors. Consistent and highly significant differences between resting and stimulated cells were demonstrable whether the 4 p28 spots were considered individually (p less than 0.0025) or in sum (p less than 0.0005). The p28 response to Con A was shown to be contained within the T lymphocyte fraction of mononuclear cells, was dose dependent over the mitogenic concentration range for Con A, and was blocked by alpha-methyl-D-mannoside, a sugar inhibitor of Con A binding. Differences between stimulated and resting cells were readily apparent within 2 hr of activation and became progressively larger with time. Protein p28 was prominently represented in plasma membranes isolated on discontinuous sucrose density gradients and was identified as a cell surface glycoprotein based on studies in which intact stimulated cells were exposed to degradative enzymes. This protein was lost from cells treated with pronase, whereas neuraminidase digestion changed the pI of p28 from a range of 5.5 to 6.2 to a range of 6.1 to 6.4 without changing the apparent m.w.

Effect of ouabain on macromolecular synthesis during the cell cycle in mitogen-stimulated human lymphocytes

Ouabain inhibited in a concentration-dependent and completely reversible way, the synthesis of DNA, RNA and protein in phytohemagglutinin and concanavalin A-stimulated human lymphocytes without affecting the uptake of nucleosides and amino acids into the cells. On the other hand, ouabain even at very high concentrations was unable to interfere with the binding of [ 3H]concanavalin A. No correlation was found between the inhibition by ouabain of macromolecular synthesis and that of K + transport. The inhibitor effect of ouabain on the stimulation of macromolecular synthesis could be partially reversed by higher concentrations of K +, due to the direct inhibition of ouabain binding. Ouabain added to the cultures at different stages of cell growth suppressed the incorporation of thymidine to various extents. Both ouabain sensitive stages fell in a period preceding the onset of mitosis and were characterized by very active thymidine incorporation. Lymphocytes were most sensitive to ouabain within the S phase. The results suggest that ouabain interferes with mitogen-triggered membrane-associated events, other than K + transport, controlling mitosis at distinct phases of the cell cycle.

Glucocorticoids and lymphocytes. II. Cell cycle-dependent changes in glucocorticoid receptor content.

To study variations in glucocorticoid receptor levels during the cell cycle, we have separated mitogen-stimulated human peripheral lymphocytes and rat lymph node cells by unit gravity sedimentation and measured glucocorticoid binding in the resultant fractions. By morphologic criteria and thymidine incorporation, the fractions were separated into populations of G0 and G1 phase and S and post-S phase cells. A 2- to 3-fold increase in glucocorticoid receptor sites per cell, for cells in the S and post-S phase over those in G0 and G1, was observed with both nonstimulated rat lymph node cell suspensions and concanavalin A-stimulated human peripheral lymphocytes. These observations together with those from other studies indicate that formation of new glucocorticoid receptors near the S phase may be a general phenomenon in proliferating cells. We propose that this increase in glucocorticoid receptors during the cell cycle may explain the increase in glucocorticoid receptors in mitogen-stimulated lymphocytes.

Inhibition of human lymphocyte transformation by oxygenated sterol compounds

Oxygenated sterol compounds (OSC), potent inhibitors of sterol synthesis in both resting and mitogen-stimulated human lymphocytes, are capable of suppressing the DNA-synthetic response of human peripheral blood lymphocytes to mitogenic lectins, anti-human thymocyte antiserum, and the mixed lymphocyte culture. The most potent OSC are 20α-hydroxycholesterol and 25-hydroxycholesterol, which inhibit DNA synthesis in mitogenstimulated lymphocytes at 7.4 and 3.9 × 10 −6 M, respectively. Lymphocytes which have been exposed to OSC for 18 hr and washed free of inhibitor are fully capable of DNA synthesis when subsequently challenged with mitogen. Suppression of DNA synthesis by OSC is not apparent during the first 24–40 hr of culture. The inhibition of lymphocyte DNA synthesis by OSC can be partially reversed by the addition of 10 −2 M mevalonate to the culture. Sterol synthesis by mitogen-stimulated lymphocytes is enhanced by culturing them in medium supplemented with lipoprotein (and cholesterol-)-depleted serum. In such medium, the 50% inhibitory doses of 25-hydroxycholesterol for suppression of both mitogen-stimulated lymphocyte DNA and sterol synthesis are approximately equal (−3 × 10 −7 M ). Sterol synthesis is a necessary, but not sufficient, part of the program of lymphocyte proliferative response to mitogens; the evidence presented suggests that lymphocytes can utilize exogenous sterol at least partially for the purpose of cell replication.

Dissociation of the proliferative response of mitogen-stimulated human lymphocytes from the release of lymphokine(S) that stimulate prostaglandin (PG) production by isolated rheumatoid adherent synovial cells (RASC)

Dissociation of the proliferative response of mitogen-stimulated human lymphocytes from the release of lymphokine(S) that stimulate prostaglandin (PG) production by isolated rheumatoid adherent synovial cells (RASC)

Cyclic guanosine 3′,5′-monophosphate and phosphodiesterase activity in mitogen-stimulated human lymphocytes

The cyclic adenosine 3′,5′-monophosphate (cyclic AMP) phosphodiesterase from human leukemic lymphocytes differes from the normal cell enzyme in having a much higher activity and a loss of inhibition by cyclic guanosine 3′,5′-monophosphate (cyclic GMP). In an effort to determine the mechanism of these alterations, we have studied this enzyme in a model system, lectin-stimulated normal human lymphocytes. Following stimulation of cells with concanavalin A (con A) the enzyme activity gradually becomes altered, until it fully resembles the phosphodiesterase found in leukemic lymphocytes. The changes in the enzyme parallel cell proliferation as measured by increases in thymidine incorporation into DNA. The addition of a guanylate cyclase inhibitor preparation from the bitter melon prevents both the changes in the phosphodiesterase and the thymidine incorporation into DNA. This blockage can be partially reversed by addition of 8-bromo cyclic guanosine 3′,5′-monophosphate (8-bromo cyclic GMP) to the con A-stimulated normal lymphocytes. These results indicate a possible role of cyclic GMP in a growth related alteration of cyclic AMP phosphodiesterase.

Correlation between cell cycle duration and RNA content.

The metachromatic fluorochrome acridine orange was used to differentially stain DNA and RNA in Chinese hamster ovary (CHO) cells and in mitogen-stimulated human lymphocytes during their progression through the cell cycle. Green and red fluorescence of individual cells, representing cellular DNA and RNA, respectively, was measured by flow cytometry. CHO cells were synchronized by selective detachment at mitosis. Their rate of progression through G1 and subsequently through S phase correlated with the content of stainable RNA. The mean duration of the G1 phase was 5.2 hours for cells with high RNA content (highest 25 percentile population) and 8.1 hours for cells with low RNA (lowest 25 percentile). The duration of S phase was 5.9 and 7.5 hours for high- and low-RNA, 25 percentile subpopulations, respectively. Lymphocytes synchronized at the G1/S boundary by hydroxyurea or 5-fluorodeoxyuridine showed extremely high intercellular variation with respect to content of stainable RNA. After release from the block they traversed S phase at rates linearly proportional to the content of stainable RNA. The duration of S phase was five hours for cells with high RNA-, six to nine hours for cells with moderate RNA- and up to 27 hours for cells with minimal RNA-content. The data suggest that the rate of progression through the cell cycle of individual cells within a population may be correlated with the number of ribosomes per cell.

Mitogenic stimulation of human lymphocytes by Pseudomonas aeruginosa galactosephilic lectins

Mitogenic stimulation of human lymphocytes by Pseudomonas aeruginosa galactosephilic lectins

Purification of a lymphokine: Osteoclast activating factor from human tonsil lymphocytes

Osteoclast activating factor is a lymphokine produced by mitogen-stimulated human lymphocytes. The current studies describe purification to essential homogeneity of the major form of osteoclast activating factor present in supernatants of phytohemagglutinin stimulated lymphocyte cultures. Preliminary chemical and biological characterization of the purified material was carried out. The active factor is a peptide which migrates in polyacrylamide gel electrophoresis as an α-2 fraction in native gels and as a 9,000-dalton species in sodium dodecyl sulfate-urea gels. The purified fraction stimulates bone resorption in vitro at doses between 0.1 and 500 ng/ml, with half-maximal stimulation at approximately 1 ng/ml.

Effects of adenine arabinoside on lymphocytes infected with Epstein-Barr virus

Low concentrations of adenine arabinoside inhibited growth of two Epstein-Barr virus producer cell lines in culture, while not significantly affecting a nonproducer cell line and a B-cell-negative line. These observations were extended to include freshly infected cells. Mitogen-stimulated human umbilical cord blood lymphocytes were unaffected by the drug at concentration levels that inhibited [3H]thymidine incorporation into the DNA of Epstein-Barr virus-stimulated cells. DNA synthesis in Epstein-Barr virus-superinfected Raji cells was also adversely affected by adenine arabinoside. However, these same low concentrations of adenine arabinoside in the triphosphate form produced less effect on DNA synthesis in nuclear systems and DNA polymerase assays than on growth or DNA synthesis in whole cells. Therefore the effects reported here of low concentrations of the drug on whole cells may be only in part related to DNA polymerase inhibition. The work reported here suggests that adenine arabinoside has multiple sites of action in infected cells.

Transport of RNA from nucleus to cytoplasm following mitogenic stimulation of human lymphocytes.

An improved method affording clean separation of nuclei from cytoplasm was utilized to study the kinetics of appearance of [3H]uridine-labelled polyadenylated (poly(A)+) and nonpolyadenylated (poly(A)−) RNA in the cytoplasm of human lymphocytes stimulated with the mitogen concanavalin A (Con A). While our previous and present studies utilizing lymphocyte preparations purified on Ficoll-Hypaque gradients had shown that the earliest observable increase in labelled total cell RNA occurred after 12 h of exposure to Con A, we were here able to detect highly significant increases in levels of uridine-labelled poly(A)− RNA in the cytoplasm of lymphocytes which had been exposed to mitogen for 6 h; these cells had been pulsed with [3H]uridine for 2 h prior to harvest. This suggested that the early effects of mitogen may be to increase processing and transport of RNA rather than to increase the production of gene transcripts. At least 13 h of exposure to Con A was required before labelled poly(A)+ RNA appeared in the cytoplasmic extracts of stimulated cells and it did not appear in those of resting cells. The lag between addition of isotope and appearance of labelled RNA in cytoplasm was inversely related to the duration of incubation of the cells with mitogen. However, once the label appeared in the cytoplasm, its rate of appearance and its final level were similar at both 12 and 36 h of incubation with Con A. These kinetic data also suggest that mitogen acts mainly to accelerate the processing and transport of RNA at least during the first 12 h of mitogenic stimulation. A concentration (0.05 μg/ml) of actinomycin D which is known to inhibit specifically the transcription of nucleolar RNA in a variety of cell types was found to affect the transcription of poly(A)+ RNA in human lymphocytes; in some experiments, specific inhibition of nucleolar RNA synthesis was observed at 0.005 μg actinomycin D per millilitre.

Prostaglandin E and mitogenic stimulation of human lymphocytes in serum-free medium

Sera used in cell cultures contain significant amount of prostaglandins (PGs). In order to vaoid any effects of contaminating PGs, the present study employed a serum-free culture medium and confirmed the inhibitory effect of prostaglandin E (PGE) on the human lymphocyte activation which had been observed previously employing a serum-containing medium. PGE 1 displayed a significantly stronger inhibitory effect on the cells than previously shown. Furthermore, reported enhancement of PGE synthesis by mitogen-activated lymphocytes could not be reproduced.

Effects of colchicine and vinblastine on the phytohaemagglutinin-induced transformation of lymphocytes.

The effects of 2 microtubular-disruptive drugs, colchicine and vinblastine, on the phytohaemagglutinin (PHA)-induced blast transformation and mitogenic stimulation of human lymphocytes were studied. Both drugs markedly inhibited cell growth and DNA synthesis and lowered the mitotic index. No microtubules were seen with the electron microscope in cells treated with PHA plus colchicine or vinblastine. Moreover, the PHA-induced development of all organelles was partially inhibited by these drugs, especially that of the Golgi complex. As compared to cells treated with PHA alone, the dictyosomes were fewer, not so clearly localized in one area of the cytoplasm, and contained a decreased number of cisternae and an increased number of vacuoles. These results indicate that cytoplasmic microtubules play an important role in the PHA-induced blast transformation and mitogenic stimulation of lymphocytes. It is suggested that the microtubules function in the structural organization of the cell and particularly the Golgi complex. In the drug-induced absence of microtubules this and other organelle systems do not respond as usual to PHA stimulation, which could largely explain the decreased cell growth. This in turn suggests that lowered mitotic activity is a result of inhibition of cell growth, as a critical amount of G1-associated cell growth is believed to be required for the initiation of DNA synthesis and thus mitosis.

Stimulation by Alcohols of Cyclic AMP Metabolism in Human Leukocytes

In this study ethanol and certain other short-chain aryl (benzyl and phenethyl) and aliphatic (methyl, propyl, butyl, and amyl) alcohols produced up to 10-fold increases in cyclic AMP (cAMP) concentrations in purified human peripheral blood lymphocytes. Ethanol concentrations as low as 80 mg/dl produced significant elevations in lymphocyte cAMP. Significant but less marked augmentation of cAMP in response to alcohols was observed in human platelets, human granulocytes, and rabbit alveolar macrophages. The mechanism of the alcohol-induced cAMP accumulation is probably secondary to membrane perturbation and consequent activation of adenylate cyclase, because ethanol directly stimulated this enzyme in lymphocyte membrane preparations but had no effect on lymphocyte phosphodiesterase activity. Lysosomal enzyme release, by phagocytosing human leukocytes, and aminoisobutyric acid transport in mitogen-stimulated human lymphocytes were shown to be inhibited by ethanol and other alcohols at concentrations which also elevate cAMP. In general, the magnitude of the inhibition of these inflammatory processes correlated with the ability of the alcohol to elevate cAMP concentrations. Lectin-and anti-thymocyte globulin-induced lymphocyte mitogenesis was inhibited or unaffected depending upon both the concentration and type of mitogenic stimulus and the concentration and type of alcohol utilized. Inflammatory mediator release from rat mast cells also was inhibited by ethanol and certain other alcohols, but whole cell cAMP was not increased. Ethanol may alter these inflammatory responses and other biologic processes at least in part by modulating cellular levels of cAMP.

Functional and Morphologic Characterization of Human T Cells Continuously Grown in Vitro

Abstract Long-term growth (now over 13 months) of thymus-derived lymphocytes from numerous normal human bone marrow and peripheral blood cell samples was accomplished by using a factor present in media obtained from mitogenstimulated human peripheral blood lymphocytes. This long-term growth could neither be initiated nor maintained by mitogens alone. All cell cultures were greater than 90% E rosette-positive, whereas the tests for B cell markers, surface IgG and IgM, and EAC rosette were routinely negative. There was no evidence for the presence of granulocytes, monocytes, and their precursors in these cultures. The E rosette-positive cells were then tested to see if they had T cell functions. PHA, Con A, and pokeweed mitogens stimulated lymphproliferative responses in these cultures comparable to those of fresh peripheral blood cells. These proliferating cells were also able to release cell mediators, such as interferon and colony-stimulating activity. Further evidence for the T lymphocyte nature of these cultured cells was obtained from one-way mixed leukocyte cultures in which these cells responded to but were unable to stimulate allogeneic cells. The functional and morphologic characteristics of these cultured cells show that these cells are T cells that grow continuously in vitro.

In vitro lymphocyte cytotoxicity II. Unstable lymphotoxins (β-LT) secreted and inactivated by mitogen-stimulated human lymphocytes

Supernatants from phytohemagglutinin (PHA)-activated human lymphocytes contain two major classes of cytotoxins (α-LT and β-LT). While α-LT appears to be stable, and the major component in 5-day culture supernatants, the majority of cytolytic activity at earlier intervals in these cultures is due to a “family” of highly unstable cytotoxins which are both secreted and destroyed at a rapid rate. The inactivation of the unstable LT molecules appears to be due to: (a) inherent instability o β-LT molecules, and (b) a lymphocyte-mediated inactivation mechanism(s) which involves serum.

E rosette formation at 37°:A property of mitogen-stimulated human peripheral blood lymphocytes

Peripheral blood lymphocytes from healthy humans formed stable E rosettes with sheep erythrocytes (SRBC) at 37°C after culture with phytohemagglutinin or the divalent cation ionophore A23187. Cells manifesting this phenomenon exhibited “blast” morphology, appeared by 16 hr of culture, increased dramatically in percentage and absolute number by 62 hr, and persisted in large numbers for the duration of culture (182 hr). Unstimulated lymphocytes formed rosettes at 4°C but not at 37°C. Increased “stickiness” due to surface-bound lectin mitogen was not the cause of rosette formation at 37°C. Formation of E rosettes at 37°C has previously been considered a property of lymphocytes less differentiated than the circulating T cell (e.g., thymocytes, leukemic lymphoblasts). The present findings indicate that this property can be “reexpressed” during blastogenesis in culture. This observation also demonstrates technical problems associated with the use of SRBC to quantitate lymphocytes with complement receptors (B cells) by the EAC rosette assay in culture. False positives resulted from 37°C E rosette formation, but this was overcome by replacing the SRBC with guinea pig erythrocytes in the EAC assay.