Arno Hausen

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.

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.

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.

Determination of neopterine in human urine by reversed-phase high-performance liquid chromatography.

Since the analysis of urinary neopterine is important in diagnosing malignancy, a method has been developed for its rapid and sensitive separation and quantitation using high-performance liquid chromatography (HPLC) on reversed phase. Eluted neopterine is monitored by fluorescence at a excitation wavelength of 353 nm and measured at 438 nm. Separation was optimized by elution with 15 mmol/l potassium phosphate (pH 6.4) at a flow-rate of 0.8 ml/min. Urinary neopterine was related to creatinine with the aim of reducing variations due to fluctuating urinary concentrations. The proposed method has good performance characteristics and is not influenced by the presence of reduced neopterine in urine. Using this HPLC method, urinary neopterine related to creatinine was determined for 148 healthy male adults (mean neopterine/creatinine 113 mumol/mol), 146 healthy female adults (mean neopterine/creatinine 140 mumol/mol) 60 healthy children (mean neopterine/creatinine 163 mumol/mol). The neopterine levels for four healthy individuals were measured daily over a period of one month.

Neopterin as a new biochemical marker for diagnosis of allograft rejection. Experience based upon evaluation of 100 consecutive cases.

The use of daily urinary neopterin evaluation to detect immunological complications has been tested in 96 consecutive cadaveric kidney recipients, three liver recipients, and one pancreas recipient. In 29 of these patients an immunologically uncomplicated posttransplant course was associated with stable or low neopterin levels, or both. In only 5% of daily determinations on these patients were increasing or high neopterin levels seen. On the other hand, major immunological complications, such as acute rejection episodes (38 cases), viral infections (17 cases), or both problems (8 cases), were preceded by increasing or high neopterin levels or both--on the average by one day. Withdrawal of cyclosporine was also found to be followed by increase of urinary neopterin levels. Neopterin evaluation enabled reliable and accurate prediction of immunological complications in 95% of patients with acute rejections and in 100% of patients with viral infections. It thus appears that daily assessment of urinary neopterin levels represents a useful tool for biochemical detection of immunological complications in allograft recipients.

Neopterin concentrations in cerebrospinal fluid and serum of individuals infected with Hiv-1

Neopterin, a biochemical marker for the activation of cell-mediated immune reactions, was determined in serum and cerebrospinal fluid (CSF) from patients infected with HIV-1. A significant correlation was found between serum and CSF neopterin concentrations, although concentrations of neopterin in serum were more closely correlated with the clinical severity of HIV-1 infection than those in CSF. However, higher CSF levels were observed in patients with neurologic/psychiatric symptoms than in unaffected patients. Also, quotients of CSF neopterin versus serum neopterin concentrations were increased, indicating intrathecal production of neopterin. Positive HIV-1 isolation from peripheral blood mononuclear cells (PBMC) was associated with higher neopterin concentrations in serum, when compared with negative HIV-1 isolation. Neopterin in CSF appears to be a suitable biochemical marker in patients with HIV-1 infection for detecting overt neurologic/psychiatric disturbances. The data suggest that in HIV-1 infected patients, cell-mediated immune reactions might be activated intrathecally and might contribute to neuropsychiatric disease.

Neopterin as an index of immune response in patients with tuberculosis.

Urinary neopterin levels were measured by high-performance liquid chromatography in 55 patients with pulmonary tuberculosis. A positive correlation between mean neopterin levels, extent and activity of disease was apparent. Neopterin levels reflect changes of disease course earlier than other currently available procedures. Neopterin levels are valuable for the management of pulmonary tuberculosis.