Hans-Peter Eck

Macrophages regulate intracellular glutathione levels of lymphocytes. Evidence for an immunoregulatory role of cysteine

Macrophages consume cystine and generate approximately equivalent amounts of acid-soluble thiol. Stimulation of macrophages with bacterial lipopolysaccharide (LPS) or tumor necrosis factor (TNF) strongly augments the amount of thiol released into the culture supernatant. Cysteine constitutes most of the acid-soluble thiol. The intracellular glutathione level and the DNA synthesis activity in mitogenically stimulated lymphocytes are strongly increased by either exogenously added cysteine, or (syngeneic) macrophages. This cysteine dependency is observed even in the presence of relatively high extracellular cystine concentration as they occur in the blood plasma. The extracellular cysteine concentration also has a strong influence on the intracellular glutathione concentration, viability, and DNA synthesis of cycling T cell clones. Moreover, the cysteine concentration in the culture medium on Day 3 and Day 4 of a 5-day allogeneic mixed lymphocyte culture (i.e., in the late phase of incubation) has a strong influence on the generation of cytotoxic T cell activity, indicating that regulatory effects of cysteine are not restricted to the early phase of the blastogenic response. The inhibitory effect of cysteine starvation on the DNA synthesis of the T cell clones and on the activation of cytotoxic T lymphocytes can be explained essentially by the depletion of intracellular glutathione, since similar effects are observed after treatment with buthionine sulfoximine (BSO), a specific inhibitor of the glutathione biosynthesis. BSO has practically no influence, however, on the N alpha-benzyloxycarbonyl Ne-t-butyloxycarbonyl-L-lysine-thiobenzyl-ester (BLT)-esterase activity and hemolytic activity of the cell lysates from cytotoxic T cells against sheep red blood cells (perforin activity). Taken together, our experiments indicate that cysteine has a regulatory role in the immune system analogous to the hormone-like lymphokines and cytokines. It is released by macrophages at a variable and regulated rate and regulates immunologically relevant functions of lymphocytes in the vicinity.

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.

Plasma glutamate levels, lymphocyte reactivity and death rate in patients with bronchial carcinoma

SummaryElevated glutamate concentrations are commonly observed in patients with advanced carcinoma, and glutamate was recently found to inhibit the membrane transport of cystine and to impair the function of macrophages and lymphocytes in vitro. We therefore investigated the possibility that elevated plasma glutamate levels may be quantitatively correlated with reduced lymphocyte reactivity and an impaired host response to the tumor. Here we report the results of a study on patients with bronchial carcinoma, which show that patients with plasma glutamate levels above 120 μM have a lower lymphocyte response to mitogens and a substantially higher death rate than those with glutamate levels below 120 μM. This correlation does not prove a causal role of glutamate, but it confirms predictions from the in vitro laboratory data.

[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.

Partial recovery of lymphocyte activity in patients with colorectal carcinoma after curative surgical treatment and return of plasma glutamate concentrations to normal levels

SummaryGlutamate was recently found to inhibit the membrane transport of cystine and to impair the function of macrophages and lymphocytes in vitro. Elevated plasma glutamate concentrations in patients with advanced carcinoma were also found to be quantitatively correlated with reduced lymphocyte reactivity in these persons. We now investigated the questions whether glutamate levels in tumor patients would decline to approximately normal levels after tumor resection and, if so, whether this would be correlated with a recovery of lymphocyte reactivity. We report that plasma glutamate levels as well as the concomitantly elevated plasma lactate levels of patients with colorectal carcinoma return to practically normal levels within 1 week after curative surgery. This is accompanied by a rapid recovery of the lymphocyte reactivity against concanavalin A. Lymphocyte responses against pokeweed mitogen and phytohemagglutinin, in contrast, remain impaired for at least 6 months, indicating that elevated glutamate levels in patients with colorectal carcinoma are associated with a long-lasting defect in the immune system.

Expression of increased immunogenicity by thiol-releasing tumor variants

Even moderate variations of the extracellular cysteine concentration were previously shown to affect T cell functions in vitro despite high concentrations of cystine. We therefore analyzed the membrane transport activities of T cells for cysteine and cystine, and the role of low molecular weight thiol in T cell-mediated host responses against a T cell tumor in vivo. A series of T cell clones and tumors including the highly malignant lymphoma L5178Y ESb and its strongly immunogenic variant ESb-D was found to express extremely weak transport activity for cystine but strong transport activity for cysteine. However, not all cells showed the expected requirement for cysteine (or 2-mercaptoethanol (2-ME)) in the culture medium. One group of clones and tumors including the malignant ESb-lymphoma did not respond to changes of extracellular cystine concentrations and was strongly thiol dependent. This group released only little acid soluble thiol (cysteine) if grown in cystine-containing cultures. The other T cell lines, in contrast, were able to maintain high intracellular GSH levels and DNA synthesis activity in cystine-containing culture medium without cystein or 2-ME and released substantial amounts of thiol. This group included the immunogenic ESb-D line. Additional thiol-releasing ESb variants were obtained by culturing large numbers of L5178Y ESb tumor cells in cultures without cysteine or 2-ME. All of these ESb variants showed a significantly decreased tumorigenicity and some of them induced cytotoxic and protective host responses even against the malignant ESb parent tumor. Taken together, our experiments suggest that the host response against a tumor may be limited in certain cases by the failure of the stimulator (i.e., the tumor) cell to deliver sufficient amounts of cysteine to the responding T cells.