Barbara Wirleitner

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.

Potential role of immune system activation-associated production of neopterin derivatives in humans

Neopterin derivatives are produced by human monocyte-derived macrophages and dendritic cells upon stimulation with interferons. Neopterin concentrations measured in urine or blood reflect activation of cellular immunity and endogenous release of interferon-γ. This review focuses on the clinical utility of measuring neopterin levels in inflammatory disease and the potential functions of neopterin as a mediator and/or modulator in the course of inflammatory and infectious processes. In vitro-studies revealed that neopterin derivatives exhibit distinct biochemical effects, most likely via interactions with reactive oxygen or nitrogen intermediates, thereby affecting the cellular redox state. Data support the hypothesis that the release of neopterin enhances the cytotoxic potential of activated macrophages and dendritic cells. In vivo, a strong correlation between neopterin levels and the severity, progression, and outcome of infectious and inflammatory diseases was found. The influence of neopterin derivatives on the cellular metabolism may provide an explanation for these clinical observations.

Monocyte-derived dendritic cells release neopterin

Increased neopterin concentrations in body fluids are found in diseases associated with activated, cell‐mediated immunity including infections, autoimmune diseases, and certain malignancies. Monocytes/macrophages are known to secrete large amounts of neopterin upon stimulation with interferon‐γ (IFN‐γ). Ontogenetically, the major part of dendritic cells (DC) belongs to the myeloid lineage. Therefore, we investigated whether cultured monocyte‐derived DC can elaborate neopterin. Cells were treated with cytokines in the presence or absence of monocyte‐conditioned medium as a maturation stimulus. DC secreted an average 3.5 nmol/l neopterin. In response to IFN‐γ, cells significantly increased their output of neopterin. In distinction to monocytes/macrophages, neopterin production in DC was highly sensitive to IFN‐α and IFN‐β. Further, lipopolysaccharides (LPS) enhanced neopterin synthesis, whereas tumor necrosis factor α, interleukin (IL)‐1β, IL‐2, IL‐10, and IL‐18 were ineffective. Simultaneously, tryptophan degradation by induction of indoleamine (2,3)‐dioxygenase (IDO) was tested in stimulated cells. Our results showed that IFN‐γ as well as LPS are inducers of IDO in DC.

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.

Enhanced Tryptophan Degradation in Systemic Lupus Erythematosus

In vitro and in vivo, tryptophan degradation was found to be associated with T cell functional loss and tolerance induction. In systemic lupus erythematosus (SLE) besides the Th2-type cytokine interleukin-10, Th1-type cytokines including interferon-gamma (IFN-gamma) are expressed especially during exacerbation of the disease. IFN-gamma stimulates the enzyme indoleamine (2,3)-dioxygenase (IDO) converting tryptophan to the metabolite kynurenine which in macrophages is subsequently degraded to other, partly neurotoxic compounds like quinolinic acid, and finally to nicrotinamides. We measured kynurenine and tryptophan concentrations in the sera of 55 SLE patients. In these patients, the concentrations of tryptophan (median, interquartile range: 53.9, 45.7-64.1 microM) were lower (p < 0.0001), and the kynurenine concentrations (2.45, 1.75-3.40 microM) were increased (p < 0.0005) compared to healthy blood donors (70.0, 63.8-80.6; 1.80, 1.45-2.27 microM, respectively). Also the kynurenine per tryptophan quotients (K/T), which allow to estimate IDO activity, were significantly higher in patients than in normals (0.043, 0.033-0.062 vs. 0.027, 0.021-0.030; p < 0.0001), indicating enhanced IDO-induced tryptophan degradation in SLE. There was no significant relationship between tryptophan, kynurenine and the SLEDAI, and also the correlation of K/T with SLEDAI was rather weak (rs = 0.243, p < 0.05). Higher K/T was found in patients presenting with serositis (p = 0.01), decrease of complement (c3, c4; p < 0.01) and blood count change (anemia, leucopenia, lymphopenia; p = 0.032) than in patients without such disease manifestations. The significant correlation found between K/T and neopterin (rs = 0.808, p < 0.001), a marker of immune activation, points to a role of immune activation to be responsible for tryptophan degradation in SLE patients.

Is hyperhomocysteinemia due to the oxidative depletion of folate rather than to insufficient dietary intake

Abstract Hyperhomocysteinemia is considered as a risk factor for cardiovascular diseases. Usually, an inverse relationship exists between homocysteine and folate levels, and supplementation with folate lowers homocysteine concentrations in patients. Therefore, hyperhomocysteinemia is mainly ascribed to the insufficient dietary intake of folate. Hyperhomocysteinemia has also been observed in infections and inflammatory diseases. Oxidative stress appears to be involved in the pathogenesis of these disorders, and associations have been found between homocysteine and e.g., neopterin concentration. Increased neopterin concentration indicates immune system activation and also allows an estimate of thus elicited oxidative stress. It may be relevant that the active cofactor, tetrahydrofolate, is very susceptible to oxidation. Immunologically induced oxidative stress could lead to folate depletion resulting in hyperhomocysteinemia. Thus, hyperhomocysteinemia in patients can be considered as an indirect consequence of hyperconsumption of antioxidant vitamins during prolonged states of immune activation.

Atorvastatin suppresses interferon-γ-induced neopterin formation and tryptophan degradation in human peripheral blood mononuclear cells and in monocytic cell lines

Inhibitors of 3‐hydroxy‐3methylglutaryl‐co‐enzyme A (HMG‐CoA) reductase, so‐called statins, are used in medical practice because of their lipid‐lowering effect and to reduce the risk of coronary heart disease. Recent findings indicate that statins also have anti‐inflammatory properties and can modulate the immune response. In vitro, we investigated the effect of atorvastatin on the T cell/macrophage system in peripheral blood mononuclear cells (PBMC) and in the human monocytic cell lines THP‐1 and MonoMac6. We monitored neopterin production and tryptophan degradation in PBMC after treatment with 10u2003µm and 100u2003µm atorvastatin in the presence or absence of 100u2003U/ml IFN‐γ, 10u2003µg/ml phytohaemagglutinin (PHA) or 10u2003µg/ml concanavalin A (ConA) and in monocytic cell lines THP‐1 and MonoMac6 with or without stimulation with 100u2003U/ml IFN‐γ or 10u2003ng/ml to 1u2003µg/ml lipopolysaccharide (LPS). In stimulated PBMC 100u2003µm atorvastatin inhibited neopterin formation and tryptophan degradation completely, whereas 10u2003µm atorvastatin was only partially effective. Also in monocytic cell lines THP‐1 and MonoMac6, atorvastatin was able to suppress IFN‐γ‐ and LPS‐induced formation of neopterin and degradation of tryptophan. Our data from PBMC agree well with previous investigations that statins inhibit T cell activation within the cellular immune response. In addition we demonstrate that atorvastatin directly inhibits IFN‐γ‐mediated pathways in monocytic cells, suggesting that both immunoreactivity of T cells and of monocyte‐derived macrophages are down‐regulated by this statin.

Neopterin and 7,8-dihydroneopterin induce apoptosis in the rat alveolar epithelial cell line L2

The neopterin derivatives, neopterin and 7,8‐dihydroneopterin, modulate the cellular oxidant‐antioxidant balance as well as the expression of the inducible nitric oxide synthase (iNOS) gene. Since apoptosis can be induced by reactive oxygen intermediates and nitric oxide (NO) we investigated whether these neopterin derivatives induce apoptotic cell death. As model we selected the rat alveolar epithelial cell line L2. 24 h incubation of neopterin (1–1000 μM) as well as 7,8‐dihydroneopterin (1–1000 μM) resulted in a significant increase of percent apoptotic cells (measured by FACS analysis). Coincubation of both pteridines with the cytomix (interferon‐γ plus tumor necrosis factor‐α) led to a significantly higher apoptosis than the cytomix alone. In contrast to the cytomix, no iNOS gene expression and no NO release could be detected after incubation with neopterin or 7,8‐dihydroneopterin. We conclude that neopterin and 7,8‐dihydroneopterin are per se inducers of apoptosis which is not mediated by nitric oxide. This may be of importance in inflammatory pulmonary diseases associated with an activation of the cellular immune system.

Tryptophan degradation increases with stage in patients with rheumatoid arthritis.

Immune system activation is known to be involved in the progression of rheumatoid arthritis (RA). The proinflammatory cytokine interferon-γ in various cells, including monocytes, induces the enzyme indoleamine (2,3)-dioxygenase (IDO), which converts tryptophan to kynurenine. In sera of 22 patients (17 women and 5 men) with RA stages 1 to 4 according to Steinbrocker, the concentrations of tryptophan and kynurenine were measured by high-pressure liquid chromatography. To estimate IDO activity, the kynurenine to tryptophan ratio (kyn/trp) was calculated. In parallel, concentrations of the macrophage activation marker neopterin were determined by enzyme-linked immunosorbent assay. Tryptophan concentrations were lower in patients with RA, and the decrease in serum tryptophan correlated with increase in stage (p<0.05). Kyn/trp correlated well with neopterin concentrations, which were elevated in most patients. Whereas higher C-reactive protein concentrations and erythrocyte sedimentation rates were observed in patients with greater disease activity, tryptophan and neopterin concentrations did not differ between patients with different subjective disease activity graded by the physician. Deficiency of the essential amino acid tryptophan in patients with RA most likely results from immune activation involved in the pathogenesis of the disease. It could also be relevant for the mood of patients, as tryptophan is the precursor of serotonin.

Rapid measurement of total plasma homocysteine by HPLC

BACKGROUNDnDetermination of plasma homocysteine has gained increasing interest during the past few years. Several HPLC methods for determination of homocysteine are available. Based on these methods, we developed a new HPLC assay for rapid and sensitive measurement of total plasma homocysteine.nnnMETHODSnAs a reducing reagent tris-(2-carboxylethyl)-phosphine is used, ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate serves as the derivatization agent. Separation is performed by reversed-phase HPLC using a precolumn and a 55-mm RP(18) cartridge; mobile phase: 0.1 mol/l KH(2)PO(4) with 5% methanol, adjusted to pH 2.7 with ortho-phosphoric acid, flow-rate 1.1 ml/min.nnnRESULTSnHomocysteine is clearly separated from other thiols, the retention time being 2.2 min, total analysis time is 6 min. Tests for linearity, recovery and precision are satisfactory, as well as the comparison with a commercial available assay method. Detection limit of the method is 0.5 micro mol/l, it could be further enhanced for measurements of even lower homocysteine concentrations in, e.g., cell culture supernatants.nnnCONCLUSIONSnThe described method is well suited for analysis of thiols in blood specimens. It is more convenient and more rapid than methods described earlier.