Gilbert Reibnegger

Neopterin as a marker for activated cell-mediated immunity: Application in HIV infection

Abstract The production of neopterin is closely correlated with activation of cell-mediated immunity. The increased neopterin concentrations appear to be produced by human macrophages specifically stimulated with gamma-interferon. As Helmut Wachter and colleagues review here, neopterin studies reveal that preactivation of cell-mediated immunity is associated with poor prognosis in cancer patients. In addition, in human immunodeficiency virus (HIV) infection, neopterin levels increase in parallel with progressive disease, are inversely correlated with CD4 + /CD8 + T-cell subset ratios and are of predictive significance.

Neopterin as Marker for Activation of Cellular Immunity: Immunologic Basis and Clinical Application

Publisher Summary This chapter focuses on the immunological basis and clinical experiences with neopterin as an indicator for activation of the cell-mediated immune system. Neopterin was isolated from larvae of bee, worker bees, and royal jelly. Comprehensive information on the chemistry of pteridines is provided. Synthetic methods for some natural pteridines are reviewed, and the reactivity of pteridines is examined. The chapter discusses some reactions that are important for measurement of neopterin in biological specimens. The low-weight metabolite neopterin is biosynthetically derived from guanosine triphosphate via 7, 8-dihydroneopterin triphosphate. In vitro, human monocytes/macrophages produce neopterin when stimulated by interferon-γ released from activated T cells. High neopterin levels were observed in clinical settings recognized to involve activation of cell-mediated immunity in acute allograft rejections, viral infections, infections by intracellular parasites and bacteria, autoimmune diseases, and certain malignancies. Studies of neopterin levels in groups at high risk for AIDS and in patients infected with HIV have demonstrated that activation of T lymphocytes and macrophages represents a crucial event for HIV production. In malignant diseases, the degree of neopterin elevation is a measure of the clinical activity and in some tumor types, serves as a measure of the extent of disease. In ovarian cancer, cancer of the uterine cervix, prostatic tumor, and carcinoma of the lung, neopterin is of predictive value.

The Role of Neopterin as a Monitor of Cellular Immune Activation in Transplantation, Inflammatory, Infectious, and Malignant Diseases

The accumulated knowledge about the organization and function of the human immune system contributes to a better understanding of the pathogenesis of most diverse disorders and is opening new avenues for therapeutic regimens. To gain further insight into the complex interactions within the components of the immune system, it has become increasingly necessary to develop rapid and simple methods to monitor the status of the immune system in patients. The determination of neopterin concentrations in human body fluids allows to investigate sensitively the cell-mediated immune status to be investigated with considerable sensitivity. In recent years it was shown that production and release of neopterin is inducible in human monocytes/macrophages by interferon gamma. Increased neopterin levels indicate endogenous formation of gamma interferon, and monitoring of neopterin levels therefore permits the activation status of the cell-mediated immune system to be examined. Neopterin concentrations in serum and in urine increase in parallel to the clinical course of infections with viruses, intracellular bacteria, and parasites. In patients with human immunodeficiency virus infection neopterin concentration in serum and urine is a significant predictor of disease progression, the statistical power being similar to CD4+ T-cell numbers. In patients with autoimmune disorders, neopterin levels correlate with the extent and the activity of the disease. Neopterin concentrations are also sensitive indicators of immunological complications in allograft recipients. In certain malignant diseases neopterin concentrations correlate with the stage of the disease and bear prognostic information. Results of neopterin measurements agree with the important role that the cellular immune system plays in these disorders.

Characteristics of interferon induced tryptophan metabolism in human cells in vitro.

Interferon-gamma-induced tryptophan metabolism of human macrophages was compared to ten human neoplastic cell lines of various tissue origin and to normal dermal human fibroblasts. Tryptophan and metabolites were determined in supernatants of cultures, after incubation for 48 h, by high-performance liquid chromatography with ultraviolet and fluorescence detection. With the exception of two cell lines (Hep G 2, hepatoma and CaCo 2, colon adenocarcinoma) in all of the ten other cells and cell lines tryptophan degradation was induced by interferon-gamma. Five of these ten formed only kynurenine (SK-N-SH, neuroblastoma; T 24, J 82, bladder carcinoma; A 431, epidermoid carcinoma; normal dermal fibroblasts), three formed kynurenine and anthranilic acid (U 138 MG, glioblastoma; SK-HEP-1, hepatoma; A 549, lung carcinoma). Only one line, A 498 (kidney carcinoma) showed the same pattern of metabolites as macrophages (kynurenine, anthranilic acid and 3-hydroxyanthranilic acid). Interferon-gamma regulated only the activity of indoleamine 2,3-dioxygenase. All other enzyme activities detected were independent of interferon-gamma, as shown by the capacity of the cells to metabolize L-kynurenine or N-formyl-L-kynurenine. Increasing the extracellular L-tryptophan concentration resulted in a marked induction of tryptophan degradation by macrophages. Contrarily, a significant decrease of the tryptophan degrading activity was observed when the extracellular L-tryptophan concentration was increased 2-fold with SK-N-SH, T 24 and J 82, 4-fold with A 431 and A 549 and 10-fold with U 138 MG and SK-HEP-1. The activity was unaffected by extracellular L-tryptophan with dermal fibroblasts and A 498. Though interferon-gamma was the most potent inducer of tryptophan metabolism, interferon-alpha and/or -beta showed small but distinct action on some of the cells. In all cells which reacted to interferon-gamma by enhanced expression of class I and/or class II major histocompatibility complex antigens tryptophan degradation was also inducible. These results demonstrate that induction of indoleamine 2,3-dioxygenase is a common feature of interferon-gamma action, that the extent of this induction is influenced by extracellular L-tryptophan concentrations and that indoleamine 2,3-dioxygenase is the only enzyme in the formation of 3-hydroxyanthranilic acid from tryptophan which is regulated by interferon-gamma.

Human macrophages degrade tryptophan upon induction by interferon-gamma

Human peripheral blood mononuclear cells, monocytes-macrophages and T-cells were stimulated with human recombinant interferon-gamma, interferon-alpha and phytohemagglutinin. The culture supernatants were analyzed for tryptophan, kynurenine, 3-hydroxyanthranilic acid, anthranilic acid and neopterin by high performance liquid chromatography. Tryptophan was decreased and the four other compounds were increased in supernatants of peripheral blood mononuclear cells activated by interferon-gamma (250 U/ml), interferon-alpha (10.000 U/ml) and phytohemagglutinin (1 microgram/ml). After splitting of peripheral blood mononuclear cells by adherence, the monocytes and macrophages but not the T-cells degraded tryptophan upon stimulation by interferon-gamma in a dose dependent manner. Supernatants of phytohemagglutinin stimulated but not of resting T-cells were found to induce tryptophan degradation by macrophages, the active principle being neutralized by an antiserum for interferon-gamma. Thus phytohemagglutinin acts by activating T-cells to release interferon-gamma which in turn induces macrophages to degrade tryptophan. In all experiments the appearance of neopterin in the culture media was correlated to the observed tryptophan degradation.

Neopterin modulates toxicity mediated by reactive oxygen and chloride species

Neopterin, a pyrazino‐pyrimidine derivative, is synthesized in excess by human monocytes/macrophages upon stimulation with interferon‐γ, a cytokine derived from activated T‐cells. Neopterin is furthermore produced constitutively. A relatively constant ratio between neopterin and its reduced form, 7,8‐dihydroneopterin, has been described in human serum. In the study presented here we tested the ability of neopterin and its reduced form to modulate the effects of cytotoxic substances like hydrogen peroxide or hypochlorous acid and N‐chloramine derivatives. We show that 7,8‐dihydroneopterin potently reduces biological and chemical effects of these substances independently from the pH value. In contrast, at slightly alkaline pH (pH 7.5) neopterin enhances hydrogen peroxide and chloramine‐T activity. This is demonstrated by increase of signal intensity in a luminol assay and also by enhancement of toxicity towards bacteria. Thus, the macrophage derived substance neopterin is able both to enhance and to reduce cytotoxicity in dependence of pH value and its oxidation state, and it may have a pivotal role in modulation of macrophage mediated effector mechanism.

Monoaminergic neurotransmitters, their precursors and metabolites in brains of Alzheimer patients

The catecholamines dopamine (DA), noradrenaline (NA) and adrenaline (A), their aminoacid precursors tyrosine (Tyr), L-3,4-dihydroxyphenylalanine (L-DOPA), two of their metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and 4-hydroxy-3-methoxy phenyl glycol (MHPG), serotonin (5-HT) and its precursor tryptophan (Trp), were measured by high pressure liquid chromatography (HPLC) with electrochemical detection in seven regions (globus pallidus, putamen, nucleus amygdalae, nucleus caudatus, substantia nigra, gyrus cinguli and raphe) of postmortem brains from eight histologically verified cases with Alzheimers disease (AD) and six histologically normal controls. Concentrations of L-DOPA, DA, DOPAC, NA and 5-HT were significantly reduced, while Tyr and MHPG concentrations were significantly increased in AD versus control patients. The concentrations of Trp and A in AD patients were not significantly different from controls. Furthermore, for most brain regions examined, significant negative correlations between Tyr and DA as well as between NA and MHPG levels were found. These data confirm and extend findings of monoaminergic systems disturbances in AD, emphasize the significance of dopaminergic deficit for AD and suggest that in pharmacotherapy of AD, attempts to restore deficits of the transmitter systems should be directed to the monoaminergic, in particular the dopaminergic system.

Immune activation and the anaemia associated with chronic inflammatory disorders

Chronic inflammatory disorders are associated with an increased risk of patients developing anaemia. There is some evidence that cytokines released during cell‐mediated immune responses are capable of inhibiting bone marrow haematopoiesis. In vitro, interferon gamma and tumournecrosis factor alpha inhibit growth of erythroid precursor cells. The mode of action of these cytokines is probably associated with their antiproliferative capacity. Decrease of serum iron and increase of storage iron in patients appears to be a consequence of the defense strategy of macrophages during long‐lasting inflammatory disorders. Decreased serum iron correlates to decreased haemoglobin concentrations. In view of this, the development of anaemia seems likely to result from the altered iron metabolism induced by stimulated macrophages. Low haemoglobin levels and associated hypoxia up‐regulate the release of erythropoietin, which can explain why increased circulating erythropoietin is usually found in patients with anaemia.

Increased concentrations of neopterin in carotid atherosclerosis

Activation of T-cells and macrophages may play a role in the pathogenesis of atherosclerosis. Therefore, serum concentrations of the immune activation markers neopterin and soluble interleukin-2 receptor were compared with routine laboratory parameters, candidate risk variables and degree of carotid atherosclerosis. Study subjects were 561 individuals (293 men and 268 women) aged between 50 and 79 years who were enrolled in a cross-sectional community based study (Ischemic Heart Disease and Stroke Prevention Study, Bruneck, Italy). Extent of carotid atherosclerosis was quantitated by an ultrasound B-mode procedure based scoring system. Detailed physical examination and quantification of laboratory and candidate risk variables were performed. By univariate as well as multivariate statistical analyses, serum concentrations of neopterin but not soluble interleukin-2 receptor were significantly higher in subjects with carotid atherosclerosis (men, 8.5 +/- 2.7 nmol/l neopterin; women, 9.6 +/- 3.3) than in those without (men, 6.7 +/- 2.3, P < 0.0001; women, 7.5 +/- 2.3, P < 0.0001). The data show that the macrophage-derived immune activation marker neopterin is closely correlated with the extent of carotid atherosclerosis. Chronic activation of immune cells, preferentially of macrophages, may play a key role in atherogenesis and/or progression of atherosclerosis.

Increased endogenous interferon-gamma and neopterin correlate with increased degradation of tryptophan in human immunodeficiency virus type 1 infection

Reduced tryptophan and increased kynurenine concentrations have been reported in patients with human immunodeficiency virus type 1 (HIV-1) infection. From in vitro data it appears that activated indoleamine 2,3-dioxygenase (IDO) is involved in this metabolic change. IDO is inducible by interferon-(IFN)-gamma. We compared serum concentrations of IFN-gamma and neopterin (the biosynthesis of which is also inducible by IFN-gamma) with serum, tryptophan and kynurenine of 42 patients with HIV-1 infection. IFN-gamma, neopterin and kynurenine levels were significantly increased compared to HIV-1 seronegative controls whereas tryptophan was significantly decreased. Various significant correlations were found between tryptophan, kynurenine, IFN-gamma and neopterin concentrations. Highest degree of correlation was found between neopterin, IFN-gamma and the kynurenine per tryptophan quotient which is the ratio between the product and the substrate concentration of IDO. The data indicate that decreased tryptophan in HIV-1 seropositives may result from chronic immune activation and can be referred to increased activation of IDO.