Regulatory Factors of Lymphocyte-Lymphocyte Interaction: I. Con A-Induced Mitogenic Factor Acts on the Late G1 Stage of T-Cell Proliferation

DNA synthesis in murine lymphocytes was augmented by a soluble factor in the supernatant of serum-free cultures of syngeneic spleen cells activated with concanavalin A (Con A). This so-called mitogenic factor (MF), which is probably identical with interleukin II, partially purified by DEAE-cellulose and Sephadex G-75 chromatography, is a fairly homogeneous molecule of 17–25 × 10³ daltons. By using partially purified MF, the role of MF in lymphocyte proliferation was investigated. Pretreatment of lymphocytes with Con A for the first 3 hr of culture, which does not in itself induce cell proliferation, markedly augmented the effect of MF. The presence of MF, however, is necessary only in a restricted stage (s) of lymphocyte proliferation. The addition and removal of MF at various times during culture showed that MF exerts its effect on a process which occurs 3–6 hr before the beginning of DNA synthesis. These results strongly suggest that MF regulates the proliferation of mitogen-stimulated lymphocytes by acting on a restricted stage (s) of the cell cycle.     

Mitogenic activity elaborated by macrophage-like cell lines acts as competence factor(s) for BALB/c 3T3 cells

The culture medium from several murine macrophage-like cell lines contained a mitogenic activity that functioned synergistically with platelet-poor plasma to induce DNA synthesis in quiescent density-inhibited BALB/c 3T3 fibroblasts. This mitogenic activity was generated by P388D₁ (and other established lines of) macrophage-like cells that were cultured either in medium alone or in medium supplemented with platelet-poor plasma. The amount of mitogenic activity produced was directly related to the length of time the macrophage-like cells were maintained in the medium. Serum-free medium conditioned by macrophage-cells did not stimulate DNA synthesis in density-inhibited 3T3 cells in the absence of plasma; however, a transient (4-hr) exposure to serum-free macrophage-conditioned medium allowed quiescent cells to respond to plasma-derived progression factors. The addition of plasma to 3T3 cells that had been treated with the macrophage-conditioned medium brought about DNA synthesis after a 12-hr lag. The mitogenic activity that was in macrophage-conditioned medium bound to DEAE-Sephadex and eluted in a single peak using a linear NaCl gradient. This macrophage-derived competence factor was not mitogenic for lymphocytes and was clearly separated by DEAE-Sephadex chromatography from the major peak of the previously described mitogenic monokine, Interleukin-I (lymphocyte activating factor).     

Mitogenic factor from inbred guinea pigs. I. Isolation of the factor

Methods are described for the reproducible elicitation of mitogenic factor (MF) from antigen-sensitive lymphocytes of inbred strains of guinea pigs. The use of inbred animals minimizes complications due to histocompatibility factors. Each of several antigens tested was effective. Mitogenic factor is released in vitro as early as 6 hr after stimulation of lymphocytes by antigen. It was obtainable from serum-free cultures in which medium RPMI-1640 was used; this should facilitate isolation of MF. The addition of 5 mMl-cysteine to cultures substantially improved the yield of MF. MF was obtained from cultures of lymph node cells of highly purified small lymphocytes, which indicates that the small lymphocyte is the source of MF in the guinea pig. It was shown that MF can induce mitosis as well as blast transformation in non-immune lymph node cells. MF from a given strain of guinea pig is capable of stimulating lymphocytes of another strain.     

Magnetic field inhibits isolated lymphocytes' proliferative response to mitogen stimulation

We aimed to find out how the exposure of isolated lymphocytes to a pulsed magnetic field (MF) affected their in vitro proliferative response to mitogenic stimulation. Cells were exposed to MF of various intensities (0.3, 0.6, and 1.2 T) at a constant frequency of 30 Hz, for a period of 60, 180, and 330 s. Then, the proliferative response of splenocytes was induced by optimal concentrations of concanavalin A (Con A; mitogenic toward T cells), bacterial lipopolysaccharide (LPS; mitogenic toward B cells), or pokeweed mitogen (PWM; mitogenic toward both populations). We found that the exposure of lymphocytes to the MF profoundly inhibited their proliferative response to mitogens. The suppressive action of the MF on B and T cell proliferation was intensified when a cooperative response of those two lymphocyte populations was simultaneously induced by PWM. The inhibitory effect of MF depended on the exposure time and MF intensity. Prolonged exposure and/or a stronger intensity of the MF weakened its inhibitory influence on the response of lymphocyte to mitogenic stimulation. The data show that an exposure to MF may influence the activity of lymphocytes in their response to mitogenic stimuli.     

Proliferation of human peripheral blood lymphocytes induced by A23187, a streptomyces antibiotic

Incubation of human peripheral blood lymphocyte cultures with streptomyces antibiotic A23187, a divalent cation ionophore, resulted in an increased rate of calcium uptake, enhanced rates of RNA and DNA synthesis, and lymphoblastic transformation. An optimal response was obtained with an initial ionophore concentration of 3–5 μM. The highest rate of thymidine incorporation was detected when the cells were labelled from the 3rd to 4th day of culture. In long-term culture the ionophore was highly toxic to the lymphocytes and optimal response was detected only if the cells were transferred to fresh medium after incubating for some hours with A23187. Both RNA and DNA synthesis, as well as calcium uptake induced by A23187 were completely inhibited if ethyleneglycol-bis-(aminoethylether)tetraacetic acid (EGTA) was present in the culture during the first 6 h of incubation. These findings support the hypothesis that calcium ion has a critical role in the mitogenic response of lymphocytes, and that calcium influx may be an important event in the initiation of proliferation. Possible mechanisms of the effects of A23187 on lymphocytes are discussed.     

Mitogenic action of tumor necrosis factor in human fibroblasts: interaction with epidermal growth factor and platelet-derived growth factor

We have previously shown that tumor necrosis factor (TNF) can increase the number of epidermal growth factor (EGF) receptors on human FS-4 fibroblasts and that this increase may be related to the mitogenic action of TNF in these cells. Here we show that TNF stimulated the growth of FS-4 fibroblasts in a chemically defined, serum-free medium in the absence of EGF. Anti-EGF receptor antibody, which blocked the mitogenic effects of EGF in FS-4 cells, did not inhibit the mitogenic action of TNF in serum-free or serum-containing medium, indicating that EGF or an EGF-like molecule was not responsible for the mitogenic effects of TNF. However, the simultaneous addition of TNF and EGF to cells grown in serum-free medium resulted in a synergistic stimulation of DNA synthesis and cell growth. The actions of TNF and EGF were also examined in growth-arrested FS-4 cells and were compared with the action of platelet-derived growth factor (PDGF). In the absence of other growth factors, TNF was a relatively weak mitogen in growth-arrested cells, compared with EGF or PDGF. Nevertheless, TNF synergized with EGF or high doses of PDGF in stimulating DNA synthesis. Furthermore, antibodies specific for TNF or the EGF receptor were used to selectively inhibit the actions of these two factors, after specific incubation periods, in growth-arrested cells treated concurrently with EGF and TNF. To produce an optimal stimulation of DNA synthesis, EGF had to be present for a longer period of time than TNF. We conclude that in their synergistic action on growth-arrested FS-4 cells, EGF was responsible for driving the majority of the cells into S phase, while TNF appeared to make the cells more responsive to the mitogenic action of EGF. The findings indicate that TNF can cooperate with, and enhance the actions of, EGF in promoting DNA synthesis and cell division.     

Proliferation and cytotoxicity of thymus lymphocytes in vitro]

Proliferative and cytollytical activity of lymphocytes was compared in lymphocyte alloimmunization of the spleen and intact thymus. The count of live cells and DNA-synthesizing cells in the thymocyte monoculture was 10--15-fold, and in mixed thymus cell culture--about 5-fold lower than the corresponding amounts of spleen cells. The index of immune thymocyte stimulation was several times greater than that of the immune cells of the spleen. The cytotoxicity peak was observed on the 4th--5th day of stimulation when the cytolytic activity of the immune thymocytes approached the action of the immune cells of the spleen. Low DNA synthesis and a marked cytotoxic activity of immune thymocytes signified that stimulation of the thymus cells in vitro permitted to obtain cell population with a high content of cytolytic T-lymphocytes.     

Cellular regulation of human lymphocyte mitogenic factor release in vitro]

Production of the mitogenic facotr by human lymphocytes stimulated with phytohemagglutinin (PHA) was investigated. Anti-PHA antibodies were used for studying the culture medium mitogenic activity. The mitogenic factor production markedly increased after lymphocyte irradiation. When macrophages were eliminated, using iron powder, mitogenic factor generation was also increased. It was demonstrated that lymphocyte irradiation and macrophage elimination stimulated the mitogenic factor production throgh different mechanisms.     

In vitro capsaicin production by immobilized cells and placental tissues of Capsicum annuum L. grown in liquid medium

Capsaicin, from green pepper fruits is used in formulated foods and in pharmaceuticals. Cell cultures of Capsicum annuum L. were obtained from seedlings on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin. In vitro-grown cells and placental tissues from fruits were immobilized in calcium alginate. Immobilized cells and placental tissues produced capsaicin which leached out into the medium. Immobilized placental tissue exhibited greater potentiality for capsaicin synthesis than immobilized cells. Production reached a level of 1345 μg capsaicin g⁻¹ of immobilized placenta on the 14th day of culture. Production of capsaicin, on replenished nutrient medium in immobilized placenta was 2400 μg on the 30th day. Ferulic acid fed to immobilized placenta at 2.5 mM level increased capsaicin production by 2-fold by the 5th day of the culture period. Of the elicitors used, curdlan was effective on capsaicin production in immobilized cells. Extracts of Aspergillius niger and Rhizopus oligosporus stimulated capsaicin production in immobilized placental tissues.     

Characterization of a human mitogenic factor (MF) released by phytohemagglutinin-activated lymphocytes.

Phytohemagglutinin (PHA)-stimulated human lymphocytes release soluble products upon subsequent incubation in fresh medium, which are strongly mitogenic for other lymphocytes. In the present investigation, some of the biochemical properties of such a factor (MF) were investigated. It was found that serum is not required in the production of MF. The mitogenic factor was stable at 56 °C for 30 min and at 80 °C for 10 min but was destroyed by treatment at 100 °C for 1 min. By gel chromatography on Sephadex the mitogenic activity was found in fractions corresponding to a molecular weight of 40,000–55,000. Moreover, isoelectric focusing indicated an isoelectric point at pH 8.0–8.5. By subjecting MF to CM-32 cellulose ion-exchange chromatography, all activity was detected in the adsorbed fractions. PHA was studied in parallel in some of the experiments. The results clearly showed that MF is distinct from PHA which induces the release of MF. MF was not adsorbed to concanavalin A-Sepharose.     

Creatine biosynthesis enzymes in the postnatal development of rats: the role of cyclo-3',5'-AMP and glucagon in the postnatal induction of liver guanidine acetate-N-methyltransferase]

The activity of enzymes of creatin biosynthesis in the rat liver and kidneys has been studied during the postnatal development. The activity of transamidinase of kidneys (E.C. 2.1.4.1.) increases gradually and linearly up to the 20th day after birth, then decreases on the 12th--25th days and increases again up to the level characteristic of the adult organism. The activity of guanidine acetate-N-methyl transferase (E.C. 2.1.1.2.) is rather high during the first days of postnatal development, then decreases and from the 15th day on increases again attaining the maximal level by the 23rd--25th day. The second period of the increase in the enzyme activity begins on the 29th--30th day of postnatal development. The results obtained suggest that the sharp increase of activity of guanidine acetate-N-methyl transferase of the rat liver during the early postnatal development is realized with the participation of cyclic 3',5'-AMP which appears to mediate the glucagon action.     

The role of monocytes in human lymphocyte activation by mitogens.

Studies were performed to determine the role of monocytes in human lymphocyte activation by mitogens. Velocity sedimentation at 1 x G in a new apparatus was utilized to obtain highly purified lymphocyte fractions (LF) nearly free of monocytes (0.02 to 0.4%) and a fraction (MF) enriched for monocytes (64 to 92%). The average peak responses of the lymphocyte fractions to phytohemagglutinin, concanavalin A, and pokeweed mitogen were 19, 10, and 9% of the responses achieved with unfractionated lymphocyte cultures containing approximately 20% monocytes. These changes were not attributable to altered dose requirements. When mitomycin-C-treated MF cells were used to reconstitute LF cultures, it was found that 4% monocytes fully restored the response to phytohemagglutinin whereas 8 to 16% monocytes were required for a normal response to the other mitogens. Higher numbers of MF cells produced supranormal responses, with 35 to 50% monocytes resulting in the optimal stimulation. Allogeneic monocytes were able to fully reconstitute the response of LF, and 2-mercaptoethanol (50 microM) was only slightly effective. In exploring possible mechanisms by which monocytes potentiate the mitogenic activity of lymphocytes, it was found that the supernatants of MF cultures could partially, but not completely, reconstitute LF responses, suggesting that contact with MF may be required for optimal effectiveness. Addition of graded numbers of monocytes to LF altered both the kinetics of the response and the peak level of proliferation. Monocyte depletion also resulted in markedly decreased survival of cultured unstimulated LF. These observations suggest a variety of possible effects of monocytes in potentiating mitogenic responses, including contact-mediated interactions with lymphocytes (possibly to present the mitogen optimally); enhancement of proliferation kinetics and the size of the responding subpopulation, and maintenance of a requisite growth factor(s) in the culture. Small differences in the monocyte content of cultured lymphocyte preparations may thus account for many of the often observed variations in mitogen responsiveness.     

Ethanol has multiple effects on DNA synthesis in fibroblasts depending on the presence of secreted growth regulators and zinc as well as the level of protein kinase C activation

Earlier we showed that in serum-starved (27 h), washed mouse fibroblasts and other cell lines 40-80 mM concentrations of ethanol (EtOH) potentiate, in a zinc (Zn2+)-dependent manner, the combined stimulatory effects of calcium (Ca2+) and insulin (Ins) on DNA synthesis. We now report that the promitogenic EtOH effects require removal of the used medium at least 6 h prior to treatments with EtOH, Zn2+, and Ins. If serum-starved (27 h) cells were continuously incubated for another 18-h period without replacing the medium, a secreted cellular factor moderately enhanced the mitogenic effect of Ins and simultaneously blocked the potentiating effect of EtOH on DNA synthesis measured during the last hour of treatments. However, the presence of Ca2+ (2.8 mM) plus Zn2+ (25 microM) or 25-300 nM phorbol 12-myristate 13-acetate (PMA) during the serum starvation period partially restored the promitogenic effect of EtOH. The PMA effect was blocked by the protein kinase C (PKC) inhibitor GF 109203X added for the second (18 h) period. Even at 300 nM, PMA failed to fully downregulate PKC-alpha, the major PKC isoform, over a 28-h period, suggesting that an activated PKC enzyme was involved in the restoration of EtOH effect. When EtOH (40-80 mM) was added for the entire serum starvation period and the incubations were continued for 18 h without removing the medium, EtOH inhibited both the combined actions of Ins and cellular factor as well as the promoting effect of newly added EtOH on Ins-dependent DNA synthesis. Coaddition of Zn2+ and PMA with EtOH prevented these inhibitory effects of EtOH. The results indicate that in mouse fibroblasts EtOH can both enhance and inhibit Ins-dependent DNA synthesis depending on the timing of EtOH treatment as well as the presence of Zn2+, cellular factors, and activators of the PKC system.     

Tumor necrosis factor stimulates proliferation of human osteosarcoma cells and accumulation of c-myc messenger RNA

The objective of this study was to establish whether human recombinant tumor necrosis factor (TNF) can significantly stimulate the proliferation of some tumor cells. Treatment with TNF had little or no effect on the growth of human tumor cells and murine NIH/3T3 cells cultured in medium with high serum concentration. Two tumor lines, SK-MEL-109 melanoma and HOS osteosarcoma cells, were adapted to grow in medium supplemented with 0.5% serum. The growth of these SK-MEL-109 cells was inhibited by TNF, but that of the HOS cells was greatly stimulated by TNF in a dose-dependent way. Treatment with 10 ng/ml of TNF resulted in a two-fold increase in the rate of cell division. This effect of TNF was also shown by measuring DNA and protein synthesis. The continuous presence of TNF was not required for its mitogenic activity on HOS cells cultured with 0.5% serum, since treatment for only one day with TNF resulted in prolonged growth stimulation. The failure of TNF to promote division of cells cultured in medium with 10% serum may possibly be explained by the presence of saturating amounts of growth factors in serum. Interferons abolished the mitogenic activity of TNF on HOS cells. Furthermore, TNF did not show synergism with insulin or epidermal growth factor in stimulating growth of these cells. The level of c-myc mRNA was increased five-fold after 30 minutes of treatment with TNF. This shows that TNF is a growth factor for HOS cells and that it induces accumulation of c-myc mRNA.     

Effect of additions of the carbon and nitrogen sources on the NADP+ and NAD+ level in the mycelium of a producing strain of P. nigricans Thom.]

The levels of NADP+ and NAD+ in the mycelium of a cultured strain of P. nigricans Thom grown on the mineral medium with 2 per cent of glucose were studied as dependent on the time and method of addition of glucose. NaNO3 or their mixture to the medium, i. e. a single addition simultaneously with the spore inoculation or on the 7th day of the culture development, divided additions on the 5th--7th days. It was shown that addition of the above sources simultaneously with inoculation affected the levels of both dinucleotides by the 5th and 8th days. Analogous additions on the 7th day in 24 hours decreased the level of NAD+, while the NADP+ concentration increased after addition of a mixture of glucose and NANO3 and remained unchanged on their separate addition. When the above sources were added in divided doses the level of NADP+ increased till the 7th day and that of NAD+ till the end of the experiment (the 8th day).     

Lack of prostaglandin involvement in the mitogenic effect of TSH on canine thyroid cells in primary culture

The production of prostaglandin E₂ (PGE₂) by cultured dog thyroid cells was high in a serum-containing medium and low in a serum-free, completely defined medium. Thyrotropin (TSH) and epidermal growth factor (EGF), two mitogenic factors for these cells, did not stimulate PGE₂ release. Indomethacin, at a concentration which completely inhibited PGE₂ production, had no effect on thyroid cell multiplication and DNA synthesis stimulated by TSH and EGF. It is concluded that cyclooxygenase products are not involved in the proliferation of canine thyroid cells and its control by TSH.     

Leukocyte migration inhibitory factor (LMIF) production in unidirectional mixed lymphocyte cultures.

Puromycin treatment of lymphocytes was used to develop a one-way test for leukocyte migration inhibitory factor (LMIF) production in the mixed lymphocyte culture (MLC) reaction. Lymphocytes incubated with this protein synthesis inhibitor induced a vigorous mediator production by nontreated allogeneic cells, being themselves unable to respond to stimulator cells. When puromycin-treated cells were stimulated with the mitogens PHA, ConA, or PWM, overall protein and DNA synthesis were significantly decreased with concomitant abolishment of LMIF production. Viability of stimulator lymphocytes was found to be essential for generation of the mediator in MLC reaction.     

Hirudin,a probe to analyze the growth-promoting activity of thrombin in fibroblasts; Reevaluation of the temporal action of competence factors

Two classes of mitogens, competence and progression factors, function synergistically to reinitiate DNA synthesis of quiescent cells in culture. Competence factors, such as Platelet-Derived Growth Factor and Fibroblast Growth Factor, deliver their mitogenic signal after only brief exposure. In contrast, progression factors, including insulin and the Insulin-like Growth Factors, are required throughout the prereplicative phase. We now report that thrombin behaves as a competence factor in Chinese hamster fibroblasts. In particular, thrombin displays a persistent effect on DNA synthesis after transient exposure (3 hours). However, use of [¹²⁵I]thrombin reveals that despite removal of thrombin from culture medium following the brief exposure, a significant amount of thrombin remains associated with cells. If cell-associated thrombin is totally removed (after a 3 hour incubation) with a specific thrombin inhibitor, hirudin, subsequent mitogenic action is totally abolished. Therefore, we propose that “competence” factors must occupy their receptors for the entire G1 period (more than 8 hours) of G0-arrested cells to trigger the mitogenic response.     

Mitogenic and cytotoxic actions of tumor necrosis factor in BALB/c 3T3 cells. Role of phospholipase activation

In addition to its cytotoxic/cytostatic action on many tumor cells in vitro, tumor necrosis factor (TNF) was recently shown to stimulate the growth of some types of cells in culture. We examined the action of TNF in BALB/c 3T3 cells which have been used extensively to study growth regulation. In subconfluent, rapidly dividing 3T3 cultures, murine (Mu) TNF was cytotoxic, while human (Hu) TNF had virtually no antiproliferative action on the cells. In contrast, in density-arrested BALB/c 3T3 cells maintained in a chemically defined, serum-free medium, MuTNF produced a dose-dependent stimulation of DNA synthesis. In stimulating DNA synthesis, MuTNF acted synergistically with both epidermal growth factor or platelet-derived growth factor. While stimulating DNA synthesis in quiescent 3T3 cultures, high doses of MuTNF (100 or 10 ng/ml) were also cytotoxic for a portion of the cells in the same cultures. Cytotoxicity was apparent 2 h after the addition of MuTNF, well before the onset of DNA synthesis. BALB/c 3T3 cell variants selected for their resistance to the cytotoxic action of MuTNF retained the capacity to respond to the mitogenic action of MuTNF, indicating that the stimulation of DNA synthesis by TNF is not a consequence of a TNF "wounding effect." Addition of TNF to density-arrested 3T3 cells resulted in the release of free arachidonic acid and palmitic acid from the cells. Quinacrine, a phospholipase inhibitor, inhibited both cytotoxicity and DNA synthesis in response to TNF, and melittin, a phospholipase activator, mimicked both the cytotoxic and mitogenic actions of TNF in quiescent BALB/c 3T3 cells. These results suggest that phospholipid breakdown is part of the essential early signal transduction events required both for the cytotoxic and mitogenic response to TNF action.