E. R. Werner

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.

2,4-diamino-6-hydroxypyrimidine, an inhibitor of tetrahydrobiopterin synthesis, downregulates the expression of iNOS protein and mRNA in primary murine macrophages

2,4‐diamino‐6‐hydroxy‐pyrimidine (DAHP), an inhibitor of GTP cyclohydrolase I, blocks the synthesis of tetrahydrobiopterin (BH4), which is a known cofactor of inducible nitric oxide synthase (iNOS). Previously, DAHP was shown to suppress the production of nitric oxide by cytokine‐activated fibroblasts, smooth muscle cells or endothelial cells which could be attributed to its function as a cofactor antagonist. Here, we demonstrate that in interferon‐γ‐activated murine peritoneal macrophages DAHP suppresses the expression of iNOS mRNA and protein in a BH4‐independent manner and, thus, acts by a novel mechanism.

A simple index relating clinical activity in Crohn's disease with T cell activation: hematocrit, frequency of liquid stools and urinary neopterin as parameters

Crohns disease is characterized by alternating acute and quiescent periods. Several indices for activity of the inflammatory process have been proposed to have criteria for prognosis of the clinical course and therapeutic efficacy. Neopterin is specifically released from human monocytes-macrophages after induction by interferon-gamma secreted from activated human T lymphocytes. Thus, urinary neopterin excretion is elevated in diseases involving activation of cellular immunity. Fifteen clinical and laboratory parameters, including urinary neopterin levels, collected from 35 visits of patients with Crohns disease, were compared using multiple linear regression analysis with a simple clinical activity index as reference. Prediction of clinical activity was best with the combination of hematocrit, weekly number of liquid stools and neopterin. A simple triple-parametric Crohns disease activity index was established on the basis of this result. Its quality was tested on independent data obtained from 25 repeat visits of 13 of these patients. A comparison with the well-known Crohns Disease Activity Index (CDAI) was performed. The results obtained with the proposed activity index were slightly better than those with the eight-parametric CDAI for the data from the first as well as from the repeat visits. We conclude that our simple index is a reliable and easily accessible measure for clinical activity in patients with Crohns disease.

An Hplc Method to Determine Tryptophan and Kynurenine in Serum Simultaneously

A method was established to measure tryptophan and kynurenine in serum simultaneously. Tryptophan is converted to kynurenine by the action of the enzyme indoleamine 2,3-dioxygenase induced by interferon-gamma (IFN-gamma). Since IFN-gamma is a Th1-cell derived cytokine, an increased tryptophan degradation rate via the kynurenine pathway can be found when the cellular immune system is activated as it is, e.g., in viral infections or in autoimmune diseases. Thus, the ratio kynurenine per tryptophan provides a possibility to estimate IFN-gamma activity in vivo and furthermore reflects the degree of immune activation. The HPLC method requires 100 microL serum. Protein is removed by trichloroacetic acid. An external albumin-based calibrator is applied, and analysis is referred to an internal standard, 3-nitro-L-tyrosine. Kynurenine and nitrotyrosine are detected via UV absorbance at 360 nm wavelength, and tryptophan is detected via its natural fluorescence at 285 nm extinction and 365 nm emission. Representative normal values of kynurenine and tryptophan were measured in the sera of 49 healthy blood donors.

Induction of Indoleamine 2,3-Dioxygenase in Human Cells in Vitro

IFN-gamma is the most effective principle responsible for IDO induction in human cells in vitro. Lymphocyte factors support the effect of other IFN species. Induction of IDO and influence of various factors on IDO is correlated to pteridine synthesis. Cooperative effects of several mediators may account for in vivo observations concerning enhanced excretion of neopterin and tryptophan metabolites in certain disease states.

Low Haemoglobin in Haemophilia Children Is Associated with Chronic Immune Activation

Anaemia is a frequent complication in patients with human immunodeficiency virus type 1 (HIV-1) infection. We tested 14 children with severe haemophilia (9 HIV-1 antibody seropositive CDC stage IIA, 5 seronegative) for haemoglobin and urinary neopterin concentrations and found a negative correlation between neopterin and haemoglobin (rs = -0.745, p = 0.007; Spearmans rank correlation). This finding suggests that chronic immune activation, possibly along with the release of specific cytokines such as interferon gamma and tumor necrosis factor alpha may be involved in the pathogenesis of anaemia.

Relationships between pteridine synthesis and tryptophan degradation.

In 1979, a fluorescent substance occurring in increased amounts in urinary specimens of patients suffering from viral infections or malignant diseases was characterized as D-erythro-neopterin (neopterin; Wachter et al. 1979). Extended studies of neopterin excretion in diseases revealed that the enhanced excretion of neopterin is coupled to clinical conditions characterized by activated cell-mediated immunity (reviewed by Fuchs et al., 1988; Wachter et al., 1989). Studies investigating the cellular background of these observations in vitro confirmed that immunological activation of the host’s peripheral blood mononuclear cells leads to release of neopterin into the culture medium. Surprisingly, large amounts of an unidentified fluorescent substance were always formed together with neopterin by the cells (Fuchs et al., 1982; Huber et al., 1983). The chemical identification of this substance as the tryptophan metabolite 3-hydroxyanthranilic acid (Werner et al., 1985a) initiated our work on the relationship between tryptophan degradation and pteridine synthesis, which is summarized in the present contribution.

Die klinische Bedeutung des Neopterin als Tumormarker

Neopterin wird in Zellen von Wirbeltieren aus Guanosintriphosphat synthetisiert. Wir konnten 1979 zeigen, das Neopterin im Harn von Patienten mit malignen Erkrankungen und viralen Infekten erhoht ist (1).