Christian Murr

Neopterin as a marker for immune system activation.

Increased amounts of neopterin are produced by human monocytes/macrophages upon stimulation with the cytokine interferon-y. Therefore, measurement of neopterin concentrations in body fluids like serum, cerebrospinal fluid or urine provides information about activation of T helper cell 1 derived cellular immune activation. Increased neopterin production is found in infections by viruses including human immunodeficiency virus (HIV), infections by intracellular living bacteria and parasites, autoimmune diseases, malignant tumor diseases and in allograft rejection episodes. But also in neurological and in cardiovascular diseases cellular immune activation indicated by increased neopterin production, is found. Major diagnostic applications of neopterin measurements are, e.g. monitoring of allograft recipients to recognize immunological complications early. Neopterin production provides prognostic information in patients with malignant tumor diseases and in HIV-infected individuals, high levels being associated with poorer survival expectations. Neopterin measurements are also useful to monitor therapy in patients with autoimmune disorders and in individuals with HIV infection. Screening of neopterin concentrations in blood donations allows to detect acute infections in a non-specific way and improves safety of blood transfusions. As high neopterin production is associated with increased production of reactive oxygen species and with low serum concentrations of antioxidants like alpha-tocopherol, neopterin can also be regarded as a marker of reactive oxygen species formed by the activated cellular immune system. Therefore, by neopterin measurements not only the extent of cellular immune activation but also the extent of oxidative stress can be estimated.

Modulation of neopterin formation and tryptophan degradation by Th1- and Th2-derived cytokines in human monocytic cells

In order to examine the regulatory effects of major Th1‐derived cytokines, such as IL‐12, and Th2 cytokines, IL‐4 and IL‐10, on the formation of neopterin and degradation of tryptophan, two metabolic pathways induced by interferon‐gamma (IFN‐γ) in human monocytes/macrophages, we investigated the human monocytic cell line THP‐1, primary human macrophages, and peripheral blood mononuclear cells (PBMC). Neopterin formation and tryptophan degradation were induced similarly by IFN‐γ in all three cell types investigated, but the effects of interleukins were different between THP‐1, primary macrophages and PBMC. In PBMC, but not in THP‐1 cells and primary macrophages, IL‐12 was found to be additive to the effects of IFN‐γ to superinduce neopterin formation and tryptophan degradation. IL‐4 and IL‐10 reduced the effects of IFN‐γ on monocytic cells, and both cytokines were additively antagonistic to IFN‐γ in PBMC and THP‐1 cells. Finally, on preincubation, but not on addition of IL‐12, the effects of IL‐4 and IL‐10 on PBMC could be abrogated, whereas no such effect was seen in THP‐1 cells. The results show that IL‐12 up‐regulates neopterin formation and tryptophan degradation by inducing additional IFN‐γ production by Th1 cells, while a direct effect of IL‐12 on monocytes/macrophages appears to be absent. Similarly, IL‐4 and IL‐10 inhibit neopterin production and tryptophan degradation in PBMC by down‐regulating Th1‐type cytokine production and possibly also via direct deactivation of IFN‐γ effects towards monocytes/macrophages. The results clearly show how Th1 cell‐mediated immunity may be up‐ or down‐regulated by endogenous cytokine production.

Immune activation and degradation of tryptophan in coronary heart disease

Background Inflammation and immune activation appear to be important in the pathogenesis of coronary heart disease (CHD). Cytokine interferon‐γ, which is released during cell‐mediated immune responses, induces indoleamine (2,3)‐dioxygenase (IDO), an enzyme degrading tryptophan to kynurenine. Therefore, immune stimulation is commonly associated with an increased kynurenine to tryptophan ratio (kyn trp−1) indicative for activated indoleamine (2,3)‐dioxygenase and a measurable decline of tryptophan.

Decreased serum tryptophan concentration predicts poor prognosis in malignant melanoma patients.

Background: Indoleamine (2,3)-dioxygenase (IDO) catalyses the initial, rate-limiting step in the degradation of the essential amino acid tryptophan. Via tryptophan deprivation, IDO activity suppresses T cell proliferation and differentiation and is thought to be a fundamental immune escape mechanism for tumor cells. Objective and Methods: To investigate the potential role of tryptophan degradation as a prognostic marker, serum tryptophan and kynurenine concentrations and the kynurenine-to-tryptophan ratio (kyn/trp) in 87 patients with malignant melanoma were compared to the course of the disease and to concentrations of the immune activation marker neopterin. Results: Compared to 49 healthy volunteers, the melanoma patients presented with lower tryptophan levels due to accelerated degradation. This was especially true for the subgroups of patients with distant metastases (p = 0.01), though not in patients with lymph node metastases or in patients who had not yet progressed. There existed a positive correlation between kyn/trp and neopterin concentrations (rs = 0.587, p <0.001). In patients who died due to dissemination of the tumor, median tryptophan concentrations were significantly decreased (p = 0.006) and kyn/trp (p = 0.03) and neopterin concentrations (p = 0.002) were higher compared to survivors. In addition, lower tryptophan concentrations as well as higher kyn/trp and neopterin concentrations predicted a shorter survival. Conclusion: Decreased serum tryptophan concentrations and elevated serum neopterin levels can be used as predictive markers for the future course in melanoma patients. Moreover, our data support previous speculations that a higher degree of IDO expression could play a crucial role for tumor progression.

Enhancement of hydrogen peroxide-induced luminol-dependent chemiluminescence by neopterin depends on the presence of iron chelator complexes

We have previously shown that neopterin, 6‐d‐erythro‐trihydroxypropyl‐pteridine, synthesized by human monocytes/macrophages upon stimulation by interferon‐γ, enhances toxicity of reactive oxygen at neutral or slightly alkaline pH (7.5), but not at acidic pH (below 6.5). In the present study, we explored in more detail the necessary requirements for neopterin to modulate the effects of hydrogen peroxide in a luminol‐dependent chemiluminescence assay. We demonstrate that neopterin enhances hydrogen peroxide effects only in the presence of iron chelator complexes like iron‐(III)‐ or iron‐(II)‐EDTA or iron‐(III)‐DTPA. Thus, iron chelator complexes together with neopterin may play an important role in macrophage‐mediated effector mechanisms.

Effect of neopterin and 7,8-dihydroneopterin on tumor necrosis factor-α induced programmed cell death

Tumor necrosis factor‐α and the formation of reactive oxygen intermediates are central mediators of apoptosis. Recent data indicated a role of neopterin and 7,8‐dihydroneopterin in oxygen radical mediated processes. We have therefore investigated the effect of neopterin‐derivatives on TNFα induced apoptosis of the monocyte‐like cell line U937. At an elevated concentration 7,8‐dihydroneopterin was found to superinduce TNFα mediated programmed cell death due to the formation of reactive oxygen intermediates. Our results imply that in combination with TNFα high concentrations of 7,8‐dihydroneopterin enhances apoptosis due to oxidative stress on cells.

Inverse association between serum concentrations of neopterin and antioxidants in patients with and without angiographic coronary artery disease

Neopterin is released from human monocyte-derived macrophages upon stimulation with interferon-gamma and is a sensitive indicator for cellular immune activation. Furthermore, reactive oxygen species (ROS) are produced in case of immune activation and inflammation. In a cross-sectional approach, plasma concentrations of neopterin and of antioxidant compounds and vitamins were compared in 1463 patients investigated by coronary angiography, which were recruited within the LUdwigshafen RIsk and Cardiovascular Health (LURIC) study. Serum neopterin concentrations were higher in patients with coronary artery disease (CAD; mean+/-S.D.: 8.7+/-7.3 nmol/L) compared to controls (7.4+/-5.0 nmol/L; Welchs t-test: p<0.001). Mean concentrations of ascorbic acid (p<0.0001), gamma-tocopherol (p<0.05), lycopene (p<0.001), lutein+zeaxanthin (p<0.05), alpha-carotene (p<0.05) and beta-carotene (p<0.05) were lower in CAD than in controls. Neopterin concentrations correlated with CAD-score (r(s)=0.156; p<0.0001) and inversely with antioxidants lycopene (r(s)=-0.277; p<0.0001) and lutein+zeaxanthin (r(s)=-0.175; p<0.0001) levels and with vitamins ascorbic acid (r(s)=-0.207; p<0.0001) and alpha-tocopherol (r(s)=-0.105; p<0.0001). The study demonstrates that higher neopterin production is associated with lower concentrations of antioxidant compounds in patients at risk for atherosclerosis. Results suggest that lower concentrations of antioxidant compounds may relate to higher grade of chronic immune activation in patients.

Association between insulin resistance, body mass and neopterin concentrations

Obesity is frequently associated with insulin resistance. Recently an important role of the cytokine tumor necrosis factor-alpha in mediating insulin resistance of obesity through its overexpression in fat tissue has been reported. In order to examine the relation of insulin resistance to obesity and to serum neopterin, as a parameter of immune activation, we studied 1234 otherwise healthy outpatients, who visited the physicians office for a medical health check-up. 7% showed elevated glucose concentrations, 34% elevated body mass indices. There were significant correlations between glucose concentrations and body mass indices and of the latter with serum neopterin concentrations. Neopterin concentrations were significantly higher in patients with elevated body mass indices (Mann-Whitney test, U = 131 358, p = 0.0003) and elevated glucose concentrations (Mann-Whitney test, U = 35 350 p =0.02). The data may indicate that moderate immune stimulation plays a role in the development of insulin resistance, and an influence of tumor necrosis factor-alpha seems to be probable.

Fructose Malabsorption is Associated with Decreased Plasma Tryptophan

Background: Fructose malabsorption is characterized by the inability to absorb fructose efficiently. As a consequence fructose reaches the colon where it is broken down by bacteria to short fatty acids, CO2, H2, CH4 and lactic acid. Bloating, cramps, osmotic diarrhea and other symptoms of irritable bowel syndrome are the consequence and can be seen in about 50% of fructose malabsorbers. Recently it was found that fructose malabsorption was associated with early signs of depressive disorders. Therefore, it was investigated whether fructose malabsorption is associated with abnormal tryptophan metabolism. Methods: Fifty adults (16 men, 34 women) with gastrointestinal discomfort were analyzed by measuring breath hydrogen concentrations after an oral dose of 50 g fructose after an overnight fast. They were classified as normals or fructose malabsorbers according to their breath H2 concentrations. All patients filled out a Beck depression inventory questionnaire. Blood samples were taken for plasma tryptophan and kynurenine measurements. Results:Fructose malabsorption is characterized by the inability to absorb fructose efficiently. Consequently fructose reaches the colon and is broken down by bacteria to short-fatty-acids, CO2 and H2. Recently we found that fructose malabsorption was associated with signs of depression. It was therefore of interest to find out whether fructose malabsorption is associated with abnormal tryptophan metabolism. Breath hydrogen concentrations were measured in 50 after an oral dose of 50 g fructose allowing to classify them as normals (n = 15) or fructose malabsorbers (n = 35). Blood samples were taken for tryptophan and kynurenine measurements. Fructose malabsorbers showed significantly lower plasma tryptophan concentrations and significantly higher depression scores compared to normals. Fructose malabsorption is associated with lower tryptophan levels which may play a role in the development of depressive disorders.

Effects of pteridines on luminol-dependent chemiluminescence induced by chloramine-T

Pteridines are ubiquitous in living organisms, but little is known about their biological functions. Different pteridines were tested for their ability to modulate luminol-dependent chemiluminescence induced by chloramine-T at pH = 7.5 and at a concentration of 100 microM for each pteridine. We observed striking differences between the compounds; whereas reduced pteridine species were generally potent scavengers, aromatic pteridines were weak to strong enhancers of the chemiluminescence. Taking into account the detailed chemical structure of the molecules, by multiple linear regression analysis a simple index was constructed that allows prediction of the effects of the different pteridines with high accuracy (linear correlation coefficient between predicted and observed values r = 0.89). The effects of different pteridines on free radical-induced chemiluminescence might bear biological significance since, for example, certain pteridines take part in enzymic reactions involving free-radical intermediates, or are related to the activation of macrophages in close relationship with the oxidative burst.