Sabine Mihm

Inhibition of HIV-1 replication and NF-kappa B activity by cysteine and cysteine derivatives.

HIV-1 proviral DNA contains two binding sites for the transcription factor NF-x B. HIV-1-infected individuals have, on average, abnormally high levels of tumour necrosis factor α (TNFα) and abnormally low plasma cysteine levels. We therefore investigated the effects of cysteine and related thiols on HIV-1 replication and NF-x B expression. The experiments in this report show that cysteine or N-acetylcysteine (NAC) raise the intracellular glutathione (GSH) level and inhibit HIV-1 replication in persistently infected Molt-4 and U937 cells. However, inhibition of HIV-1 replication appears not to be directly correlated with CSH levels. Cysteine and NAC also inhibit NF-x B activity as determined by electrophoretic mobility shift assays and chloramphenicol acetyl-transferase (CAT) gene expression under control of NF-x B binding sites in uninfected cells. This suggests that the cysteine deficiency in HIV-1-infected individuals may cause an over-expression of NF-x B-dependent genes and enhance HIV-1 replication. NAC may be considered for the treatment of HIV-1-infected individuals.

Modulation of lymphocyte functions and immune responses by cysteine and cysteine derivatives

Mitogenically stimulated human peripheral blood lymphocytes and T cell clones were found to have weak membrane transport activity for the disulfide cystine but strong membrane transport activity for the thiol amino acid cysteine. Cysteine, however, is represented at the lowest concentration among all protein-forming amino acids in the blood plasma. Complementary laboratory experiments have shown that the cysteine supply is indeed limiting for important lymphocyte functions. Proliferative responses of mitogenically stimulated lymphocytes and T-cell clones and the activation of cytotoxic T cells in allogeneic mixed lymphocyte cultures are strongly influenced by small variations in the extracellular cysteine concentration even in the presence of relatively high and approximately physiologic concentrations of cystine. Cysteine can be substituted by N-acetylcysteine but not by cystine. The more detailed analysis revealed that the extracellular supply of cysteine influences strongly the intracellular level of glutathione (GSH) and also the activity of the transcription factor NF kappa B that regulates the expression of several immunologically relevant genes. In vitro experiments including double-chamber experiments with macrophages and lymphocytes revealed, moreover, that cysteine plays an important role as a regulatory mediator between these cell types. The cysteine supply is impaired directly or indirectly in several pathologic conditions that are associated with immunodeficiencies, including the acquired immune deficiency syndrome (AIDS). Cysteine or cysteine derivatives may therefore be considered for the treatment of patients with HIV-1 infection.

Interleukin-2 mRNA expression, lymphokine production and DNA synthesis in glutathione-depleted T cells

The stimulation of DNA synthesis in lymphocyte populations was previously shown to depend strongly on the intracellular glutathione (GSH) level. Since T cell growth is known to depend on interleukin 2 (IL-2), the experiments in this report were designed to determine whether intracellular GSH depletion may inhibit IL-2 production or the IL-2 dependent DNA synthesis. Our experiments revealed that IL-2 production and DNA synthesis of mitogenically stimulated splenic T cells have indeed different requirements for GSH. The addition of relatively high concentrations of GSH (5 mM) to cultures of concanavalin A (Con A)-stimulated splenic T cells was found to augment strongly the DNA synthesis but inhibited the production of IL-2. Moderate intracellular GSH levels, however, are apparently not inhibitory for IL-2 production, since intracellular GSH depletion by cysteine starvation or by graded concentrations of DL-buthionine sulfoximine (BSO) had virtually no effect on IL-2-specific mRNA expression and the production of T cell growth factor (TCGF). The DNA synthesis activity, in contrast, was strongly suppressed after GSH depletion with either method. As in cultures of splenic T cells, GSH depletion had no substantial effect on the induction of IL-2 mRNA and TCGF production in several mitogenically stimulated T cell clones. Taken together, our experiments suggest that complex immune response may operate best at intermediate GSH levels that are not too high to inhibit IL-2 production but sufficient to support DNA synthesis.

ROLE OF CYSTEINE AND GLUTATHIONE IN HIV INFECTION AND CANCER CACHEXIA : THERAPEUTIC INTERVENTION WITH N-ACETYLCYSTEINE

Publisher Summary Massive loss of skeletal muscle mass (cachexia) is sign of HIV infection, sepsis, and trauma, and a cause of death among cancer patients. In HIV infection, the immunological dysfunction develops progressively into a severe condition called acquired immunodeficiency syndrome. Increasing evidence suggests that an abnormal cysteine and glutathione metabolism plays a decisive role in the development of catabolic conditions and associated immunological dysfunctions. This chapter gives a conceptual overview; the chances for a therapeutic intervention with cysteine derivatives such as N-acetylcysteine (NAC) and the methodological aspects. The agenda is to identify abnormal biochemical patterns in different catabolic diseases and conditions, abnormal biochemical parameters that are significantly correlated with mortality, disease progression, and weight loss, to prove cause-and-effect relationships by experimental intervention, to develop and optimize interventive strategies that may be suitable for clinical therapy, and to test new therapeutic strategies in clinical trials. Discussed are the elevated venous plasma glutamate levels as universal marker for catabolic and precatabolic conditions—evidences for an impairment of muscular membrane transport activities, abnormal cysteine catabolism and glutathione level in skeletal muscle tissue, and the pathological significance of decreased membrane transport activity and elevated venous plasma glutamate levels. There is discussion on “Push” and “Pull” mechanisms in catabolic and precatabolic processes, decreased plasma cystine levels and evidence for “Push” and “Pull” mechanisms in sepsis, HIV Infection, and other malignant diseases, abnormally low plasma cystine and glutamine levels in patients with chronic fatigue syndrome (CFS), and the hypothetical mechanism of the “pull” condition—evidence for an abnormal cysteine and glutathione metabolism in liver. Noted are the immunological implications—cysteine deficiency and immunological dysfunction, abnormal glutathione levels in HIV Infection, and redox regulation by glutathione and glutathione disulfide. Also, there are details on the therapeutic intervention with a cysteine derivative: effects of long-term treatment with N-acetylcysteine.

[13] Effect of reactive oxygen intermediates and antioxidants on proliferation and function of T lymphocytes

Publisher Summary The importance of thiols and especially of cysteine and glutathione to lymphocyte function has been known for several years. The extracellular concentrations of thiols have a profound effect on B and T cell responses in vitro . There are also strong indications that cyst(e)ine may play a limiting role for the immune system in vivo . Certain lymphocyte functions, however, are potentiated by hydrogen peroxide or other reactive oxygen intermediates. The intact immune system thus appears to require a delicate balance between prooxidant and antioxidant conditions. This is maintained by a limited and well-regulated supply of cysteine. The pathological changes in cellular cysteine supply that are seen in persons infected with the human immunodeficiency virus (HIV) are associated with an immunological disorder. This chapter describes several experimental approaches that are being used to determine the intracellular concentrations, redox states, the functions of cysteine and glutathione, and the role of functionally related metabolites.

Regulation of T-cell functions by L-lactate

Lactate is a product of glycolytically active macrophages. After stimulation with concanavalin A accessory cell-depleted splenic T-cell populations were found to produce only minute amounts of T-cell growth factor (TCGF); but substantial amounts of TCGF were produced if the cultures were supplemented either with splenic adherent cells or with lactate but not with interleukin-1 (IL-1). IL-1 was capable, however, of supporting TCGF production by the thymoma subline EL4-6.1. TCGF production in cultures of accessory cell-depleted splenic T-cell populations was demonstrable with 10(-3) M L-lactate, and optimal responses (plateau level) were obtained with 4-6 X 10(-2) M L-lactate. Cultures of macrophages were found to accumulate up to 5 X 10(-2) M lactate. Our experiments indicate, therefore, that lactate serves as a regulatory signal by which macrophage-like accessory cells enhance helper-T-cell functions. Lactate is apparently not the only mediator of accessory cell function since plateau levels of TCGF production were markedly lower with lactate than with splenic accessory cells; but L-lactate was found also to determine the magnitude of T-cell-mediated immune responses in vivo and in cultures of unfractionated lymphocyte populations. The production of interferon in accessory cell-depleted and concanavalin A-treated T-cell cultures, however, was not significantly affected by lactate. Concanavalin A-stimulated splenic T-cell populations were found to consume glucose rapidly and to release lactate into the supernatant. This indicates that the cells contain more lactate and pyruvate than they can utilize by their respiratory metabolism. The administration of external lactate or pyruvate was found to inhibit the utilization of glucose by the mitogenically stimulated T cells.

[59] Oxidant-antioxidant status in human immunodeficiency virus infection

Publisher Summary The hallmarks of the immunopathology of human immunodeficiency virus (HIV) infection are the selective and progressive CD4+ T cell depletion and the cellular dysfunction, which is observed even prior to the depletion of CD4+ cells and which also affects other immunologically relevant cells including CD8+ T cells and B cells. This chapter summarizes data suggesting that the acquired immunodeficiency syndrome may be (at least partly) the consequence of a virus-induced cysteine deficiency. Elevated plasma glutamate levels aggravate the cysteine deficiency, because glutamate inhibits competitively the membrane transport of cystine. Standard laboratory techniques are available to determine the plasma amino acid levels and the intracellular glutathione level either in unfractionated preparation of peripheral blood mononuclear cells and monocytes or at the level of individual cells with a cytofluorometric technique. The established cysteine and glutathione deficiency of HIV-infected persons suggests the possibility that the intracellular oxidant/antioxidant status may be disturbed. Glutathione disulfide assays are available, but, in view of the generally much lower glutathione disulfide level, these assays would require many more cells.

Regulation of cytotoxic T-lymphocyte activation by l-lactate and pyruvate

The activation of cytotoxic T lymphocytes (CTL) in vivo was found to be strongly augmented by two injections of 0.2 ml 1 X 10(-1) M pyruvate in spite of the relatively high concentration of glucose (approximately 10(-2) M) in the blood. The repeated injection of 1 X 10(-1) M L-lactate, in contrast, was found to suppress cytotoxic responses in vivo. The activation of CTL and DNA synthesis in mixed lymphocyte cultures, on the other hand, was found to be suppressed by pyruvate (1 X 10(-2) M), and was substantially augmented by 1 X 10(-2) M L-lactate. The glucose concentration in the culture medium was also approximately 10(-2) M. Taken together, these results suggest that the utilization of glucose is relatively ineffective and that the respiratory metabolism is a more effective source of energy during the early T cell activation. The results suggest also that the aerobic glycolysis of macrophages and their release of L-lactate may contribute to their function as accessory cells in immune responses. The differences between the in vivo and in vitro results are discussed.

[23] Thiols and the immune system: Effect of N-acetylcysteine on T cell system in human subjects

Publisher Summary Interest in the role of thiols in the immune system was originally focused on the need to optimize lymphocyte cultures for studying other regulatory components of the immune system in vitro . This chapter describes some experimental approaches that are being used to determine the role of cysteine and cysteine derivatives in the immune system. The chapter evaluates the therapeutic effects and potential side effects of cysteine derivatives. The T cell system is profoundly influenced even by relatively moderate changes in the extracellular cysteine concentration and the intracellular glutathione and glutathione disulfide levels. The limiting role of cysteine is mainly the consequence of the weak cystine transport activity of lymphocytes and the relatively low extracellular concentrations of reduced cysteine in blood plasma or cell cultures. Activated macrophages have been shown to deliver substantial amounts of reduced cysteine and to increase the intracellular glutathione levels of activated T cells in their vicinity.

Downregulation of T cell growth factor production by ornithine decarboxylase and its product putrescine: D,L-α-difluoromethylornithine suppresses general protein synthesis but augments simultaneously the production of interleukin-2

Treatment of EL-4 lymphoma cells with tetradecanoylphorbol-acetate (TPA), a well-known activator of protein kinase C, induces the production of the T cell growth factor interleukin-2 (IL-2) and the expression of IL-2-specific mRNA within 4-8 h. This system is an ideal model for studies on the induction of a differentiated function in a homogeneous lymphoid cell population by a defined signal. TPA induces also an increase of ornithine decarboxylase (ODC) activity and elevates the intracellular concentrations of putrescine and polyamines within 4-8 h. A similar increase of intracellular putrescine and polyamine concentrations can be achieved by administration of 2 mM putrescine to the culture medium. However, putrescine cannot induce the production of IL-2 in the absence of TPA and cannot reconstitute the IL-2 production in cultures with PGE2 or cyclosporine A, i.e., two well-known immunosuppressive substances which inhibit ODC activity. Putrescine has rather a counter-regulatory effect as concluded from the observation that the TPA-induced TCGF production and IL-2-specific mRNA expression are augmented (superinduced) by the ODC inhibitor D,L-alpha-difluoromethylornithine (DFMO) and again suppressed after the administration of putrescine or polyamines to DFMO-treated cultures. The glycolytic activity, general protein synthesis [( 3H]leucine incorporation), and the cell cycle progression from G2/M to G1, in contrast, are inhibited by DFMO and reconstituted by putrescine. This demonstrates that the cells are able to sacrifice to a large extent several vital functions including their general protein synthesis and to devote themselves at the same time to a fulminant production of their functionally most relevant protein IL-2. This process is downregulated by ODC and its product putrescine. A correlation between increased IL-2 production and accumulation of cells in the G2/M phase was also observed in cultures treated with hydroxyurea or with a combination of amethopterin and adenosine.