C.Robin Benzie

The mechanism of action of the immunosuppressive drug FK-506.

The novel immunosuppressive drug FK-506 inhibits the induction of lymphocyte proliferation in vitro by mitogenic combinations of phorbol esters and calcium ionophores. Early events inducible by phorbol esters alone are unaffected, while changes induced by calcium ionophores alone are completely suppressed by as little as 0.1 nM FK-506. The event in lymphocyte activation inhibited by FK-506 is completed early in the response. Its completion requires the provision of a Ca2+ signal and concurrent activation of protein kinase C and is accelerated as a function of the strength of protein kinase C activation.

Rapid loss of sensitivity of mitogen-induced lymphocyte activation to inhibition by cyclosporin A

The ability of the immunosuppressive drug cyclosporin A (CS-A) to inhibit the activation of lymphocytes by phytohaemagglutinin (PHA) and concanavalin A (Con A) is progressively lost over the 8-hr period following mitogen addition. This process is dependent on the presence of Ca2+ in the culture medium and is complete at a time when activation still requires the continued presence of the mitogen. While inhibition by CS-A is reduced to some extent by lymphokines produced by mitogen-activated cultures, the initial loss of sensitivity to CS-A is too complete and too rapid to be accounted for in this way. We conclude that CS-A inhibits an early Ca2+-dependent step in mitogen-induced activation that is not in itself sufficient to commit the cells to initiate proliferation, but is required for later steps in the activation process, including lymphokine production.

T lymphocyte activation through the C28 pathway is insensitive to inhibition by the immunosuppressive drug FK-506

Nanomolar concentrations of the novel immunosuppressive drug FK-506 inhibit the proliferation of human T lymphocytes in vitro induced by mitogenic lectins or by monoclonal antibodies directed against the CD3 or CD2 surface antigens. However, the alternative pathway of T lymphocyte proliferation induced by monoclonal antibodies specific for CD28 together with phorbol esters is unaffected by FK-506.

Early events during the activation of human lymphocytes by the mitogenic monoclonal antibody OKT3

The effects of the mitogenic monoclonal antibody OKT3 on the metabolic changes preceding DNA synthesis during the activation of human peripheral blood mononuclear cells were compared with those induced by PHA. The aspects studied included uridine transport, the incorporation of inositol into phospholipids, Na+-dependent amino acid uptake, and protein synthesis. All four parameters were increased in response to the ligation of the T lymphocyte receptor recognized by OKT3. These changes were apparent as early as the corresponding changes induced by PHA. However, the increases in uridine uptake and inositol incorporation were disproportionately reduced when compared to those caused by PHA, and no evidence of high-dose inhibition was seen in cells activated by OKT3. This suggests that at least some lectin-induced changes in metabolism are mediated through additional mechanisms, probably involving distinct receptors.

Inhibition of initiation of protein synthesis in rabbit reticulocyte lysates by a factor present in lymphocyte cytoplasm.

Unstimulated lymphocytes from peripheral blood have only a low rate of protein synthesis, but this is greatly increased well before the initiation of DNA synthesis when the cells are induced to proliferate by mitogens [l] . The increase in protein synthesis appears to be due to an increased rate of translation of preexisting mRNA by preexisting ribosomes [2-61, caused by an increase in the rate of the initiation step [6-91. Studies with cell-free protein synthesizing systems have suggested that the low rate of initiation in unstimulated lymphocytes is due to low activity of one or more of the protein initiation factors [lo] . A similar restriction in the rate of initiation occurs in haemindeprived reticulocytes. Detailed study of this system has shown that the rate of initiation is limited by a translational inhibitor which accumulates in the absence of haemin [ 11 ,I 21. The inhibitor appears to be a protein kinase, which phosphorylates and inactivates the initiation factor eIF-2 [13,14]. Protein kinases with similar activity have been found in the cytoplasm of other mammalian cells [15-l 71, but their physiological significance is uncertain. We report here that lymphocyte cytoplasm contains an active translational inhibitor which inhibits the initiation of protein synthesis in reticulocyte lysates. The specific activity of this inhibitor is decreased after activation of lymphocytes by phyto-

Activation of T lymphocytes by 12-O-tetradecanoylphorbol-13-acetate is resistant to inhibition by cyclosporin A

Cultured T lymphocytes from pig blood can be activated by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Activation is additive with that induced by the mitogenic lectin phytohaemagglutinin (PHA). Activation by TPA differs from that induced by PHA or other mitogenic lectins in that it is not inhibited even by high concentrations of the immunosuppressive drug cyclosporin A (CS-A). Neither co-culture of lymphocytes with PHA and TPA nor addition of culture supernatants from TPA-stimulated cultures affected the sensitivity to CS-A of the response to PHA.

The role of the transferrin receptor in lymphocyte activation

Blockade of the transferrin receptors whose expression is induced in lymphocytes incubated with the mitogenic lectin phytohaemagglutinin (PHA) does not affect the initial stimulation of protein synthesis but does strongly and progressively inhibit the subsequent induction of DNA synthesis. When the effects of transferrin receptor blockade on the induction of the enzymes uridine kinase (whose induction begins early in G1 phase of the cell cycle) and thymidine kinase (whose induction is closely associated with DNA synthesis) were examined, both enzymes were found to be induced normally. This indicates that the function of the transferrin receptor is directly to provide a component essential for DNA synthesis itself (probably iron) rather than to act as the receptor for a general signal required to initiate entry into S-phase.

Direct effects of 1,3-diaminopropane on reticulocyte lysate protein synthesis

The precise functions of polyamines in mammalian cells have not yet been established, but the strong association between the rate of cell growth and proliferation and the rates of putrescine and spermidine synthesis suggests that they have an important regulatory role [ 11. The short half-lives of some enzymes required for polyamine synthesis, in particular the first enzyme of the pathway ornithine decarboxylase, and the consequent very rapid and extensive variation in its activity, reinforce this suggestion. Specific inhibitors of the production of polyamines would be of great value in further elucidating their functions, and one drug which has been widely used in this respect is the putrescine analogue 1,3-diaminopropane [2-171. Diaminopropane does not directly affect the activity of isolated ornithine decarboxylase, but greatly reduces the activity of the enzyme in the intact animal or cell [2,8,10,11]. As the enzyme synthesis can also be inhibited, apparently by a feedback repression mechanism, by low concentrations of its natural product putrescine [4,18,19], it seemed likely that diaminopropane was acting by a similar mechanism, although substantially higher concentrations of the analogue were required. However, it has been inferred from studies with virus-infected baby hamster kidney cells that the effects of diaminopropane may be due at least in part of a direct effect on cellular protein synthesis [20]. The dramatic effects on ornithine decarboxylase may thus be a function of its unusually rapid turnover rate rather than, or in addition to, any specific inhibition of its synthesis by a feedback repression mechanism. Here we report that diaminopropane, at concentrations within the range normally used, does indeed have a direct inhibitory effect on the rate of protein synthesis by a cell-free system from rabbit reticulocytes. The effect of diaminopropane is not due to any antagonistic effect against naturally occurring polyamines, but is simply a cation effect consequent upon the high levels used. In the absence of normal divalent cations, diaminopropane will itself support protein synthesis in this system. These results lend strong support to the doubts about the specificity of diaminopropane as an inhibitor of polyamine synthesis raised in [20].

REGULATION OF PROTEIN SYNTHESIS DURING LYMPHOCYTE ACTIVATION BY PHYTOHAEMAGGLUTININ

Publisher Summary This chapter describes the regulation of protein synthesis during lymphocyte activation by phytohaemagglutinin. The rate of protein synthesis begins to increase early after the addition of mitogens to lymphocyte cultures, and prior to the induction of DNA synthesis, it rises to about ten times the rate found in unstimulated lymphocytes. This increase does not depend on the synthesis of new ribosomes, and there is no change in the rate at which individual protein molecules are synthesized. Studies with lymphocyte cell-free protein synthesizing systems have shown that the low rate of protein synthesis in unstimulated lymphocytes is not because of a lack of mRNA but because of a shortage of the initiation factors required for its translation. One of the initiation factors involved is eIF-2, but a further factor or factors necessary for a later step in the initiation sequence are also affected. A similar pattern of inhibition of the initiation sequence can be induced in reticulocyte cell-free systems by the addition of small quantities of lymphocyte cytoplasm, which suggests that these cells contain a translational inhibitor that prevents protein synthesis by inactivating initiation factors.

Initiation reactions in the mRNA-dependent reticulocyte lysate

Reticulocyte lysates depleted of mRNA by digestion with micrococcal nuclease still show an unexpectedly high rate of formation of 80 S initiation complexes. Formation of these complexes is sensitive to all inhibitors of the normal protein synthesis initiation process tested. Such lysates contain high concentrations of mRNA fragments which can be utilized for initiation, with which exogenous mRNA must compete. As a consequence of this competition, mRNAs that are weak initiators may be translated poorly by this system even at low exogenous mRNA concentrations.