Effect of polymyxin-B on T-lymphocyte protein synthesis

The role of protein kinase-C (PK-C) protein phosphorylation on the mitogen triggered responses of T-lymphocytes was examined by observing the effect of polymyxin-B (an inhibitor of PK-C) on mitogen induced protein and DNA synthesis. Polymyxin-B inhibited 3H-thymidine incorporation by PHA activated T-lymphocytes over a range of PHA concentrations. 3H-leucine incorporation by PHA activated T-lymphocytes was inhibited by polymyxin-B in a dose dependent manner. A partially purified PK-C fraction from polymyxin-B treated PHA activated T-lymphocytes demonstrated less than 25% of the phosphorylating activity of untreated lymphocytes. These results suggest that protein synthesis during the T-lymphocyte activation process is dependent on PK-C activity.     

Protein synthesis and ribosome activation during the early stages of phytohemagglutinin lymphocyte stimulation

The rate of protein synthesis by lymphocytes increases linearly from 3 h after the addition of phytohemagglutinin (PHA). There is a linear increase in the percentage of active ribosomes between 3 and 12 h of lymphocyte culture with PHA. Thus, an important early event during the activation of lymphocytes by mitogen is a stimulation in the rate of initiation of protein synthesis.     

Stimulatory effect of Bestatin,a new specific inhibitor of aminopeptidases,on the blastogenesis of guinea pig lymphocytes

A potent protease-inhibitor of Actinomycetes origin, Bestatin. which is of dipeptide nature and inhibits aminopeptidase B and leucine-aminopeptidase competitively, strongly stimulates blastogenesis of small lymphocytes triggered with polyclonal mitogen. such as phytohemagglutinin (PHA), concanavalin A (Con A), pokeweed mitogen (PWM) and lipopolysaccharide of Escherichiae coli (LPS), whereas it inhibits DNA synthesis of normal resting lymphocytes. The stimulatory effect is non-selective with respect to the category of small lymphocytes, i.e. T- and B-lymphocytes, but strikingly selective with respect to the stage of blastogenesis: the stimulation is greatest at a relatively early stage, diminishes as mitogen-activation proceeds, and is not appreciable at a later stage of lymphocyte blastogenesis.The pattern of Bestatin stimulation on lymphocyte blastogenesis is specific for the mitogen used: in T-lymphocyte activation with PHA or Con A, the stimulation first increases and then decreases with increase in mitogen concentrations, whereas in B-lymphocyte activation with LPS, with increasing concentrations of the mitogen, the stimulation increases to a plateau at approximately 100 μg/ml of mitogen. The optimum concentration of Bestatin was found to be approximately 50 μg/ml (0.16 mM) for either PHA or Con A activation, and 50 to 75 μg/ml for B-cell activation with LPS. Bestatin must remain in cultures of T- and B-lymphocytes with polyclonal mitogens for at least about 24 and 16 hr, respectively, to exert its stimulatory effect on blastogenesis.Biochemical results, together with those from autoradiographic analyses, indicate that Bestatin increases the number of blastoid-transformed lymphocytes with polyclonal stimulants. It is suggested that aminopeptidases, possibly located at the cell surface, may play a role in the control of lymphocyte activation during immune responses.     

Mitogen induction of deoxyuridine triphosphatase activity in human T and B lymphocytes

Deoxyuridine triphosphatase (dUTPase; deoxyuridine diphosphohydrolase; EC 3.6.1.23) activity during mitogen stimulation was investigated in human T-cell and B-cell enriched mononuclear leucocyte fractions as well as in a mixed lymphocyte population. The dUTPase activity was very low in the resting peripheral blood lymphocytes. A remarkable enhancement of enzymatic activity was observed when cells were stimulated with different mitogens; T-cells and non-separated lymphocytes with phytohaemagglutinin, and the B-cell enriched fraction with pokeweed mitogen. There was a positive correlation between dUTPase activity and the enhancement of macromolecule synthesis (protein and RNA). In particular, a highly significant correlation was observed between dUTPase activity and DNA synthesis in the three human lymphocyte populations studied. This supports the view that the enzyme dUTPase may have a significant role in cellular proliferation. The physiological role of the enzyme is discussed.     

Posttranslational formation of hypusine in a single major protein occurs generally in growing cells and is associated with activation of lymphocyte growth

Growing lymphocytes perform a novel chemical modification of a single protein (Hy+: approximately 18 kd, pI approximately 5.1), resulting in the formation of the unusual amino acid, hypusine (N epsilon-[4-amino-2-hydroxybutyl]lysine). This posttranslational event occurs only following activation of lymphocyte growth. Hypusine formation increases at a rate parallel to protein synthesis during the first 24 hr of growth stimulation, beginning before 6 hr of growth. At all times, hypusine is restricted primarily to the single protein, Hy+. In resting cells, the unmodified substrate protein, Hy0, is continuously synthesized and maintained in a steady-state pool of significant size. In several other cell lines, hypusine formation was also observed in a single protein of approximately 18 kd, pI approximately 5.1, indistinguishable electrophoretically from the lymphocyte protein. Thus Hy+ is a ubiquitous protein showing significant conservation among divergent species. Maintenance by resting lymphocytes of a pool of unmodified protein and early activation during growth of the hypusine-forming enzyme system suggest that this posttranslational modification may be of importance to lymphocyte activation.     

Stimulation of S-adenosylmethionine synthetase in human lymphocytes by streptococcal M protein

The effects of the specific antigen M5 protein of group A streptococci on AdoMet synthetase activity and AdoMet levels in peripheral blood (PB) lymphocytes were studied and were compared with the effects of the nonspecific polyclonal T cell mitogen PHA. M5 protein stimulated AdoMet synthetase activity, whereas PHA had a biphasic effect with an early inhibitory effect and a later stimulatory effect on AdoMet synthetase activity. S-Carbamyl-L-cysteine (SCC), an inhibitor of human lymphocyte AdoMet synthetase, reduced AdoMet levels and inhibited the blastogenic response of PB lymphocytes to both M5 protein and PHA. Inhibition of the response to M5 protein was stronger than that to PHA. However, the inhibitory effects of SCC were totally reversible by washing the cells. It is our hypothesis that such differences in the biochemical events triggered by specific antigen as opposed to a polyclonal mitogen may determine the direction of the functional differentiation of T lymphocytes.     

Spatial redistribution of ribosomal chromatin in the fibrillar centres of human circulating lymphocytes after stimulation of transcription

We have studied the distributional changes of the completely extended ribosomal chromatin present in the fibrillar centres of resting human lymphocytes after phytohemagglutinin (PHA) treatment. In thin sections of resting lymphocytes selectively stained for DNA, the extended non-nucleosomal chromatin was located in a solitary, large agglomerate which corresponds to the solitary, large fibrillar centre observed in uranium-lead-stained sections. At 20 h after PHA stimulation the ribosomal chromatin agglomerate appeared to be fragmented into smaller agglomerates which correspond to numerous fibrillar centres surrounded by a thick rim of dense fibrillar component. The mean area of ribosomal chromatin agglomerates from resting lymphocytes was found to be 0.772 mu 2 + 0.125 SD, whereas in stimulated lymphocytes it was found to be 0.184 mu 2 + 0.052 SD. At 20 h after PHA treatment ribosomal RNA (rRNA) synthesis was 8-fold greater than the control value, whereas DNA synthesis had not started. These results indicate that ribosomal chromatin of resting lymphocyte fibrillar centres contains transcribable sequences, temporally not expressed.     

Culture conditions affecting induction and release of lymphocyte produced proliferation inhibitory factor (PIF)

Studies on the factors affecting the production of a proliferation inhibitory factor (PIF) by human lymphocytes are presented. Maximal PIF production occurred with mitogen stimulation of blood lymphocytes cultured at 1 × 10⁶/ml. Optimal cultures contained 10% fetal calf serum, but PIF could be produced in the absence of serum, and after only a 6-hr pulse exposure to PHA. PIF production was found to correlate with lymphocyte activation in response to the mitogen PHA but was not related to lymphocyte proliferation (DNA synthesis). Inhibitory activity could be detected as early as 3 hr after mitogen addition, long before DNA synthesis occurs. The mitogens Con A and PWM initiated different intensities of DNA synthesis in these cultures, but similar quantities of PIF. Antigenic stimulation of sensitive human peripheral lymphocyte populations resulted in the release of PIF. Cells from donors that gave a strong positive skin test to tuberculin (PPD) responded in tissue culture to PPD by producing PIF, while the cells from skin test negative donors did not. A small quantity of PIF was also evident in the supernatants from cultures with no known stimulus (“unstimulated”), this was found to result from activation of the lymphocytes by nonlymphoid elements and by fetal calf serum. An investigation of the PIF-producing capabilities of other lymphoid tissues showed that lymph node cells produced this humoral factor, whereas thymus cells did not. Thymus cell supernatants, in fact, were found to contain an extremely labile cytotoxin which degraded rapidly upon storage.     

Mechanism of lymphocyte activation. II. Requirements for macromolecular synthesis in the production of lymphokines

Reversible inhibitors of protein synthesis, cycloheximide and puromycin, and an irreversible inhibitor of RNA synthesis, actinomycin D, were employed to study the kinetics and types of macromolecular synthetic events required for the production of migration inhibitory factor (MIF) and macrophage activating factor (MAF) by Con A-stimulated lymphocytes. Reversible inhibition of protein synthesis during the first 2 hr of stimulation completely inhibited MIF and MAF production. The same treatment, performed 4 hr after the beginning of the stimulation, had no effect. When the inhibitors of protein synthesis were left in the cultures, a block of lymphokine production was observed when the drugs were added at 6 hr as well as at time 0. In contrast, irreversible inhibition of RNA synthesis at 6 hr was ineffective and only treatment at the beginning of culture blocked lymphokine production. These data suggest that a critical protein is synthesized during the first few hours of stimulation, which is required for subsequent production of lymphokines. After this special early requirement, however, continued protein synthesis is needed for lymphokine production. In contrast, the RNA required for MIF and MAF production seemed to be completely synthesized within 4 to 6 hr of stimulation. The possibility that suppressor macrophages inhibit lymphokine production via modulation of macromolecular synthesis is discussed.     

Suppression of lymphocyte stimulation by anterior hypothalamic lesions in the guinea pig

Anterior hypothalamic lesions in the guinea pig inhibited lymphocyte stimulation in whole blood cultures with the antigen tuberculin and with the mitogen phytohemagglutinin (PHA) and suppressed the delayed cutaneous hypersensitivity response to tuberculin. The lesions did not affect the stimulation of purified lymphocytes with either tuberculin or PHA. The anterior hypothalamic lesions had no effect on the absolute number of T and B lymphocytes.     

Cell-mediated immunity to influenza virus antigen and mitogen in guinea pigs: measurement with a semimicro protein synthesis assay

A rapid and precise method for the assay of cell-mediated immune response basing on protein synthesis stimulation of mitogen-activated guinea pig lymphocytes is modified in a way that enables the study of virus-immunological problems. When used as a micromethod it has the following advantages over conventional methods: short-term cell culture, need of low quantities of cells and rapid preparation of great numbers of samples for radioactivity measurements. In this study we report the results of comparative experiments on measuring lymphocyte stimulation after addition of PHA and stimulation of sensitized lymphocytes following contact with homologous influenza virus antigen in vitro. The most important reaction parameters are as follows: 5-6 . 10(5) spleen lymphocytes/microculture in microtiter plates, use of Eagles's MEM cell culture medium without leucine, supplemented with HEPES buffer and 10% autologous guinea pig serum; optimum lymphocyte stimulation by addition of 0.5 microliter PHA or 0.1-1.0 microgram virus protein/ml; immuno-stimulation by PHA can be measured in vitro already after 6 h and by influenzavirus antigen already after 24 h.     

Induction of human gamma interferons by a mitogen derived form Mycoplasma arthritidis and by Phytohemagglutinin: differential inhibition with monoclonal anti-HLA.DR antibodies

The peripheral blood lymphocytes from eight healthy volunteers produced significant amounts of interferon (IFN) when co-cultured with a recently discovered mitogen preparation derived from cultures of Mycoplasma arthritidis (MAS). Maximum levels of 281 to 2187 U were reached after 3 to 4 days of incubation. The IFN was characterized as IFN gamma on the basis of pH and heat lability, and neutralization with anti-human IFN gamma but not anti-IFN alpha. A monoclonal antibody to HLA.DR determinants suppressed IFN induction and lymphocyte proliferation when co-cultured with MAS but was not effective against PHA-induced IFN and proliferation; PHA-induced proliferation was enhanced in the presence of the antibody. Anti-HLA.DR-mediated inhibition of lymphocyte functions, however, was not specific for MAS inasmuch as viral-induced IFN was also suppressed, as was Con A-induced proliferation. These and other studies suggest that the cellular requirement for T cell activation by MAS, PHA, and Con A may all be distinct.     

INHIBITORY EFFECTS OF MITOGENS ON ADENOIDAL LYMPHOCYTES IN VITRO

Cultures of human adenoidal lymphocytes exposed briefly to either phytohemagglutinin (PHA), Staphylococcus filtrate (Staph-F), concanavalin-A(Con-A), or pokeweed mitogen (PWM) incorporate increased amounts of thymidine earlier than replicate cultures exposed continuously to the mitogens. These effects can begin in the first 24 hr of culture and are seen maximally between 36 and 72 hr. Once a blastogenic response is established, PHA or PWM can diminish that response. Inhibition with PWM requires that the initial stimulation was with this mitogen, while PHA can inhibit blastogenesis to both PHA and PWM-stimulated cells. Because these mitogens can have a paradoxical effect on adenoidal lymphocytes, being capable of both initiating and inhibiting DNA synthesis, this phenomena should be kept in mind when such systems are utilized for the evaluation of antigens and drug effects.     

Lymphocyte lactate dehydrogenase isoenzymes in association with depressed mitogen responsiveness

Purified lymphocyte preparations from cancer patients were less responsive to the mitogen phytohaemagglutinin (PHA) than were lymphocytes from healthy donors as measured by [3H]-thymidine uptake over periods in culture up to 96 hours. The uptake of radiolabel was paralleled by total cellular lactate production. The isoenzymic composition of lactate dehydrogenase (LD) in lymphocytes from healthy individuals was altered following PHA stimulation with increasing proportions of LD-1 and LD-2 throughout the culture period. This phenomenon was markedly reduced in lymphocytes from cancer patients. This defect in lymphocytes from cancer patients is thought to reflect an impaired capacity to accomplish an early mitogen-induced enhancement of glucose metabolism, which is a prerequisite for lymphocyte proliferation.     

Early synthesis of specific cytoplasm proteins is correlated with the rate of exit of lymphocytes from the resting state

We investigated the initiation of synthesis of proteins in human lymphocytes exposed to the mitogen phytohemagglutinin (PHA) for 6 h. Radiolabeled proteins in three subcellular fractions, cytoplasmic, nuclear salt wash, and nuclear, were separated on polyacrylamide gels. Compared with cells incubated for the same time in the absence of PHA only two cytoplasmic proteins of Mr 51 and 60 kd showed increased synthesis in a dose-dependent fashion. Synthesis of the 60-kd protein shows the strongest correlation with rate of entry into the first S phase and with rate of cellular aggregation. Thus, the 60-kd protein appears to be a major early response-associated protein for entry of lymphocytes into the first S phase after PHA stimulation.     

PHA刺激对淋巴细胞修复DNA损伤的影响

本文报道PHA刺激对淋巴细胞DNA修复的影响的实验结果。以254nm波长的UV照射细胞(30J/m~2)引起DNA损伤,以[~3H]-TdR掺入实验测定非程序DNA合成,用超微量法测定细胞的NAD~+含量,并以[~(35)S]-蛋氨酸掺入,聚丙烯酰胺凝胶电泳及放射自显影术测定蛋白质生物合成,其结果如下: (1)在被PHA转化的淋巴细胞内非程序DNA合成,随PHA刺激的时间加长而增高;PHA处理淋巴细胞42小时,合成的速率约增加4倍;(2)在转化的淋巴细胞内,非程序DNA合成及程序DNA合成都被N-乙基马来酰亚胺(一种DNA聚合酶α的抑制剂)抑制,表明在DNA修复过程中DNA聚合酶α可代替DNA聚合酶β发挥作用; (3)UV照射后,被PHA刺激的淋巴细胞内NAD~+含量大约减少43.2％,而对照淋巴细胞内NAD~+的含量只减少25％,似乎说明PHA刺激能促进淋巴细胞内的P-ADP-核糖化作用;(4)在受PHA刺激72小时的淋巴细胞内有多种蛋白质合成,这些细胞在UV照射后以含10μg/ml嘌呤霉素的培养基培养,则非程序DNA合成被明显抑制(P<0.01),这提示DNA修复是一需要蛋白质合成的过程。此外,在受UV照射后10-45小时的淋巴细胞内,诱导产生一种分子量大约34000道尔顿的蛋白质。 上述结果表明,当PHA使淋巴细胞从静止状态转化为增殖状态时,有多种酶被诱导。由于这些酶,如DNA聚合酶α及P-ADP-核糖聚合     

Inhibition of T-lymphocyte activation by amiloride

The T-lymphocyte activation process involves a series of coordinately coupled biochemical events occurring in response to antigen or mitogen. These events have not been completely characterized. The present studies investigate the mechanism of protein synthesis during the initial phase of T-cell activation. Among the early biochemical changes, induction of protein synthesis was observed as early as 10 minutes after mitogen stimulation of T-lymphocytes. This early protein synthesis was inhibited by cycloheximide but was insensitive to actinomycin-D, indicating the presence of preformed mRNA in resting lymphocytes. Since early protein synthesis parallels the increase in protein kinase C activity in activated T-lymphocytes, these two biochemical events may be related. In the present report, amiloride, an inhibitor of Na+/H+ antiport and protein kinase C, significantly inhibited [3H]leucine and [3H]thymidine incorporation in a dose-dependent manner into phytohemagglutinin (PHA)-stimulated T-lymphocytes. Furthermore, when T-lymphocytes were stimulated by phorbol myristate acetate, a known activator of protein kinase C, a similar inhibition of protein and DNA synthesis by amiloride was observed. The partially purified cytosol fraction isolated from PHA-activated T-lymphocytes showed a 75% decrease in protein kinase C-mediated [32P] incorporation from ATP in the presence of 100 microM amiloride. These results suggest that the T-cell activation process following exposure to mitogens involves early protein synthesis, which may be mediated by protein kinase C.     

Mechanism of activation of protein synthesis initiation in mitogen-stimulated T lymphocytes

The pronounced stimulation of protein synthesis in T lymphocytes in response to mitogens is partly due to increased cell size and hence ribosome number. There is also a large increase in translation rate per ribosome as a result of an increased rate of initiation. In response to mitogen, levels of both eukaryotic initiation factor (eIF)-2 and guanine nucleotide exchange factor, GEF, increase in parallel with ribosomes which is consistent with a general increase in the translational machinery but cannot explain the increase in activity per ribosome. However, as total eIF-2 accumulates, the ratio of phosphorylated eIF-2 alpha (eIF-2(alpha P] to eIF-2 alpha decreases. Further, the levels of eIF-2(alpha P) and GEF in resting T lymphocytes are similar. As eIF-2(alpha P) inhibits GEF by effectively sequestering the exchange factor in an inactive 1:1 complex, the level of GEF available for protein synthesis initiation must be very low in resting cells. Hence, as GEF is synthesized and rises above the level of eIF-2(alpha P), there will be a disproportionate increase in GEF available for initiation compared with the increase in total GEF. This increase in available GEF is probably great enough to support the increase in translation rate per ribosome as well as the increase in ribosome number.     

Progressive loss in vitro immune response with tumor growth.

Peripheral blood lymphocytes from rats carrying a transplantable hepatoma were cultured in the presence of phytohemagglutinin (PHA), concanavalin A (ConA) or dextran sulfate (DS) at various times after tumor cell inoculation or after its surgical removal. Mitogen-induced lymphocyte transformation, measured by tritiated thymidine incorporation, declined as the tumor size increased, especially when cells were cultured in autologous serum. The response to PHA and ConA declined prior to the response to DS. This inhibition could not be removed by extensive washing of the cells, alteration of serum concentration, time of incubation or mitogen dose. Culture for 24 hr prior to the addition of high doses of mitogen resulted in partial restoration of the PHA and ConA, but not DS, responses. Previously inhibited responses also returned when the tumor was surgically removed. Spleen cells from animals with large tumors were also inhibited.