Chapter 1 Introduction
1.2 The immune response of monocytes to malaria
1.2.7 Neopterin, a marker of inflammation
The role of IFN-γ in communications between T cells and macrophages with the subsequent release of neopterin, make plasma measurements of neopterin an ideal method for measuring immune activation within a patient (Wachter et al., 1989). IFN-γ results in a rapid and sustained increase in neopterin levels in plasma (Muller et al., 1991). Neopterin can be easily measured in plasma, urine and cerebrospinal fluid by high performance liquid chromatography because of
its high fluorescence (Rippin, 1992; Werner et al., 1987a; Werner et al., 1987b), although many clinical laboratories also use immuno-based methods such as enzyme-linked immunosorbent assay to measure neopterin (Westermann et al., 2000).
Neopterin levels rise rapidly in parallel with C-reactive protein levels in response to an infection well before a patient becomes sero-positive. Monitoring neopterin levels in plasma can be used to assess the efficacy of treatment used in a range of infections such as malaria (Awandare et al., 2006a; Reibnegger et al., 1984), human immunodeficiency virus (Baier-Bitterlich et al., 1996a; Fuchs et al., 1988) and tuberculosis (Fuchs et al., 1984b; Yuksekol et al., 2003). As elevated neopterin levels are indicative of inflammatory conditions, plasma neopterin concentrations can serve as a primary screen for blood donations (Zangerle et al., 1992). The measurement of plasma neopterin levels has also been used in the study and management of cancer (Fuchs et al., 1984a; Reibnegger et al., 1991), autoimmune disease (Leohirun et al., 1991; Reibnegger et al., 1986; Schroecksnadel et al., 2003) and transplant patients (Margreiter et al., 1983; Reibnegger et al., 1991) where an increase in plasma or urine levels gives clinicians adequate warning of allograft rejections enabling them to alter immunosuppressant treatment. Serum neopterin has also been found elevated in patients with unstable angina and acute myocardial infarction (Schumacher et al., 1992; Schumacher et al., 1997; Tatzber et al., 1991).
1.2.7.1 Biosynthesis of neopterin and 7,8-dihydroneopterin
The biosynthesis of neopterin is similar to the pathway leading to tetrahydrobiopterin, an essential cofactor of several mono-oxygenases and inducible nitric oxide synthases (Gorren and Mayer, 2002). Refer to Figure 1.6. GTP-cyclohydrolase I (EC 3.5.4.16), an enzyme up- regulated by IFN-γ, catalyses the breakdown of guanosine triphosphate to 7,8-dihydroneopterin triphosphate. In non-primate macrophages, for example, mouse macrophages, 7,8- dihydroneopterin triphosphate is converted by 6-pyruvoyltetrahydropterin synthase (PTPS) in an Mg2+-dependent step to form 6-pyrovoyltetrahydropterin. In the final step, yielding tetrahydrobiopterin, sepiapterinreductase catalyzes the NADPH-dependent reduction of 6- pyruvoyltetrahydropterin. Human and primate monocytes and macrophages have lower levels of 6-pyruvoyltetrahydropterin synthase activity (Schoedon et al., 1987; Werner et al., 1990), hence activation of GTP- cyclohydrolase 1 leads to an accumulation of 7,8-dihydroneopterin
N N H
N N
N H2
O
O O
OH OH P O
OH P O O
OH
OH OH O
O P
N N H
N
N H O
N H2
OH
OH
O OH O
O
P O
OH O
P OH
OH O P
N N H
N
N H O
N H2
OH
OH OH
N N H
N
N O
N H2
OH
OH OH N
N H
N H
N H O
N H2
O
O
CH3
N N H
N H
N H O
N H2
OH
OH
CH3
GTP-cyclohydrolase 1 Guanosine triphosphate
7,8-Dihydroneopterin triphosphate 7,8-Dihydroneopterin
Phosphatases
Oxidation
Neopterin 6-Pyrovoyltetrahydropterinsynthase
6-Pyrovoyltetrahydropterin
Sepiapterinreductase
5,6,7,8-Tetrahydrobiopterin
Figure 1.6. Synthesis of 5,6,7,8-tetrahydrobiopterin and neopterin. The synthesis of neopterin has similarity to the pathway leading to 5,6,7,8-tetrahydrobiopterin. GTP- cyclohydrolase 1, a key enzyme in the process, catalyses the conversion of guanosine triphosphate to 7,8-dihydroneopterin triphosphate, which in the 5,6,7,8-tetrahydrobiopterin synthetic pathway, is converted to 6-pyrovoyltetrahydropterin in a step catalysed by 6- pyrovoyltetrahydropterinsynthase. The transformation of 6-Pyrovoyltetrahydropterin to 5,6,7,8- tetrahydrobiopterin is catalysed by sepiapterinreductase. In human and primate monocytes and macrophages, there are very low activities of 6-pyrovoyltetrahydropterinsynthase, resulting in an accumulation of 7,8-dihydroneopterin triphosphate that is cleaved by non-specific phosphatases to 7,8-dihydroneopterin which is then oxidized in a non-enzymatic step to neopterin (Gieseg et al., 2008; Murr et al., 2002).
triphosphate which is released as 7,8-dihydroneopterin due to the action of intracellular phosphatases. 7,8-Dihydroneopterin diffuses out of the activated macrophage into the intercellular spaces and finally into the plasma. Some of the 7,8-dihyroneopterin is oxidized to 7,8-dihydroxanthopterin by reactive oxygen species. The main reaction generating neopterin from 7,8-dihydroneopterin is oxidation by hypohalous acids such as hypochlorous acid (HOCl) (Widner et al., 2000). Neutrophils, and possibly macrophages release large amounts of HOCl during inflammation (Chisolm et al., 1999; Schraufstatter et al., 1990) suggesting that neopterin measured in plasma comes from sites of inflammation where HOCl is released.
1.2.7.2 Functions of 7,8-dihydroneopterin and neopterin
A clear biological function for neopterin and its derivatives is not completely understood at this stage, but they do appear to play a role in oxidative stress. Neopterin was found to enhance chloramine-T and H2O2-mediated chemiluminescence in vitro (Weiss et al., 1993). This suggested neopterin had pro-oxidant properties which was further substantiated by neopterin enhancing tyrosine nitration (Widner et al., 1998) and low density lipoprotein-(LDL)-oxidation (Herpfer et al., 2002) by peroxynitrite. Moreover, neopterin was also found to augment H2O2-, hypochlorite-, or chloramine-T-mediated toxicity against bacteria (Horejsi et al., 1996; Weiss et al., 1993) and induce apoptosis in vascular smooth muscle cells and the alveolar type II-like epithelial cell line L2 (Hoffmann et al., 1998; Schobersberger et al., 1996). However, neopterin also displayed anti-oxidant effects: in the absence of iron, neopterin was a potent scavenger of H2O2-induced chemiluminescence (Murr et al., 1994); and neopterin also suppressed NADPH- oxidase in macrophages stimulated with phorbol-12-myristate-13-acetate, and thus decreased the generation of superoxide anions (Kojima et al., 1992).
7,8-Dihydroneopterin displays anti-oxidant properties at low concentrations. It inhibited: the luminescence signal from superoxide and hydrogen peroxide (Shen, 1994); tyrosine nitration by peroxynitrite (Widner et al., 1998); and metal ion and aqueous peroxyl radical (2,2’- azobis(amidinopropane)dihydrochloride)-mediated LDL oxidation (Gieseg et al., 1995). 7,8- Dihydroneopteirn also suppressed the toxicity of H2O2, hypochlorite, or chloramine-T against bacteria (Horejsi et al., 1996; Weiss et al., 1993). Furthermore, micromolar concentrations of 7,8-dihydroneopterin inhibited cellular damage to red blood cells and human monocytic U937 cells from a range of oxidants such as hydrogen peroxide, hypochlorite, aqueous peroxyl radicals, nitric oxide and direct plasma membrane oxidation by ferrous ions (Gieseg et al.,
2000; Gieseg et al., 2001a; Gieseg et al., 2001b). These findings have led to the hypothesis that 7,8-dihydroneopterin secreted by IFN-γ-stimulated macrophages protects these antigen- presenting cells from oxidants encountered in the inflammatory site (Duggan et al., 2002;
Gieseg et al., 1995; Kojima et al., 1992; Schroder et al., 1987).
However, at high concentrations, 7,8-dihydroneopterin acts as a pro-oxidant and induces apoptosis in the presence of TNF-α in the human neuronal cell line (NT2) (Spottl et al., 2000), astrocytic and microglial cell lines (Speth et al., 2000) and the rat pheochromocytoma cells (PC12) (Enzinger et al., 2002b) by the formation of ROI. At concentrations below 300 µM, 7,8- dihydroneopterin diminished TNF-α-induced programmed cell death in U937 cells, whereas 5 mM 7,8-dihydroneopterin enhanced the effect of TNF-α on apoptosis (Baier-Bitterlich et al., 1995). Likewise with Jurkat T cells, 7,8-dihydroneopterin only induces apoptosis above 1 mM (Baier-Bitterlich et al., 1996a; Wirleitner et al., 1998; Wirleitner et al., 2001) via the redox- sensitive Bcl-2 pathway (Enzinger et al., 2002a).
Neopterin and its derivatives have also been shown to play a role in cell signalling. Micromolar levels of both neopterin and 7,8-dihydroneopterin increased intracellular calcium levels in human-derived monocyte-like THP-1 cells (Woll et al., 1993) and nanomolar levels of neopterin effectively inhibited ATP-induced calcium release from alveolar epithelial cells (Hoffmann et al., 2002). Micromolar concentrations of neopterin were also reported to cause cardiac contractile dysfunction in isolated perfused rat hearts (Balogh et al., 2005; Margreiter et al., 2000).
Neopterin and 7,8-dihydroneopterin were found to interfere with intracellular signalling pathways that are influenced by oxidative stress. Neopterin and 7,8-dihydroneopterin activated the redox-sensitive transcription factor nuclear factor-κB (NF-κB) in Jurkat cells (Baier-Bitterlich et al., 1997) and murine vascular smooth muscle cells (Hoffmann et al., 1996). In vitro, neopterin inhibited hypoxia-induced erythropoietin gene expression and formation in HepG2 cell cultures (Schobersberger et al., 1995b) and hypoxically perfused isolated rat kidneys (Pagel et al., 1999). Neopterin also stimulated cytokine-inducible nitric oxide synthase gene expression in rat vascular smooth muscle cells (Schobersberger et al., 1995a). Both neopterin and 7,8-dihydroneopterin together with cyclic-GMP induced the redox-sensitive proto-oncogene c-fos in NIH 3T3 fibroblasts (Uberall et al., 1994) and neopterin also enhanced the cell damage caused by UV-A irradiation of Β-16 melanoma cells (Kojima et al., 1995).