Chapter 3 Effect of antimalarial drugs, haemozoin ( β -haematin), Plasmodium
3.1 Introduction
Neopterin is biosynthetically derived from guanosine triphosphate (GTP) (Murr et al., 2002) by GTP-cyclohydrolase 1, which catalyzes the cleavage of the purine to produce 7,8- dihydronoepterin triphosphate (Werner et al., 1990). In most cells, like fibroblasts and endothelial cells, 7,8-dihydroneopterin is used to synthesize 5,6,7,8-tetrahydrobiopterin which is an essential cofactor for mono-oxygenases and inducible nitric oxide synthases (Gorren and Mayer, 2002). However, due to the lower levels of 6-pyruvoyltetrahydropterin synthase in primate and human monocytes and macrophages, activation of GTP-cyclohydrolase 1 results in an accumulation of 7,8-dihydroneopterin triphosphate which is converted by intracellular phosphatases into 7,8-dihydroneopterin (Schoedon et al., 1987). 7,8-Dihydroneopterin diffuses out of the activated macrophage into the intracellular spaces and finally into the plasma. The main reaction generating neopterin from 7,8-dihydroneopterin is oxidation by hypochlorous acid (HOCl) (Widner et al., 2000) which is released from neutrophils and macrophages during inflammation (Chisolm et al., 1999; Schraufstatter et al., 1990). The neopterin:7,8- dihydroneopterin ratio of 1:2 is nearly constant in urine, serum or cerebrospinal fluid, whereas higher neopterin:7,8-dihydroneopterin ratios are found in arterial blood in comparison to venous blood (Weiss et al., 1992b).
Neopterin derivatives are produced by human monocyte-derived macrophages (Huber et al., 1984) and dendritic cells (Wirleitner et al., 2002) when stimulated with the cytokine, interferon- gamma (IFN-γ), that is released from activated T helper (Th) cells subtype 1 (Romagnani, 1991). As these cells promote an immune response mediated by cytotoxic T cells, increased production of neopterin in body fluids can be used to monitor cell-mediated immunity (Wachter et al., 1989). 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). However, many clinical laboratories also use immuno-based methods such as enzyme-linked immunosorbent assay (ELISA) to measure neopterin (Westermann et al., 2000). The levels of neopterin in body fluids are elevated in infections such as malaria (Awandare et al., 2006a; Reibnegger et al., 1984), human immunodeficiency virus infection (Baier-Bitterlich et al., 1996b; Fuchs et al., 1988) and tuberculosis (Fuchs et al.,
1984b; Yuksekol et al., 2003), malignancies (Fuchs et al., 1984a; Reibnegger et al., 1991), autoimmune diseases (Leohirun et al., 1991; Reibnegger et al., 1986; Schroecksnadel et al., 2003), allograft rejection (Margreiter et al., 1983; Reibnegger et al., 1991), cardiac and renal failure (Roccatello et al., 1992), coronary artery disease (Schumacher et al., 1992; Tatzber et al., 1991) and myocardial infarction (Schumacher et al., 1997). Neopterin measurements not only provide insight into the cell mediated immune response but also allow monitoring and prognosis of disease progression.
The role of 7,8-dihydroneopterin and neopterin during the inflammatory process remains poorly understood and controversial. Neopterin acts as a pro-oxidant, enhancing oxidant damage (Murr et al., 1994; Wede et al., 1999; Weiss et al., 1993) and triggering apoptosis in a number of different cell types (Baier-Bitterlich et al., 1995; Hoffmann et al., 1998; Schobersberger et al., 1996). In contrast, 7,8-dihydroneopterin acts as an antioxidant at low concentrations. It reacts with and neutralizes a range of reactive oxygen species including hypochlorite, nitric oxide and peroxyl radicals, thus protecting lipoproteins and various cell types including macrophages and red blood cells (Baird et al., 2005; Firth et al., 2008; Gieseg and Cato, 2003; Gieseg et al., 2001a; Gieseg et al., 1995; Gieseg et al., 2001b). 7,8-Dihydroneopterin inhibited direct oxidation of plasma membranes of U937 cells by ferrous ions (Gieseg et al., 2001b). Thus, it has been suggested that IFN-γ-stimulated macrophages synthesize 7,8-dihydroneopterin to protect these antigen presenting cells from the oxidants encountered within an inflammatory site (Duggan et al., 2002; Gieseg et al., 1995; Kojima et al., 1992). However, there are instances where high concentrations of 7,8-dihydroneopterin have pro-oxidant properties (Baier-Bitterlich et al., 1996a; Baier-Bitterlich et al., 1995; Enzinger et al., 2002b; Speth et al., 2000; Spottl et al., 2000; Wirleitner et al., 1998; Wirleitner et al., 2001).
Neopterin and its derivatives have also been reported to play a role in cell signalling. Neopterin and 7,8-dihydroneopterin were found to increase intracellular calcium concentrations (Hoffmann et al., 2002; Woll et al., 1993), activate redox-sensitive transcription factor nuclear factor-κB (Baier-Bitterlich et al., 1997), inhibit hypoxia-induced erythropoietin gene expression (Pagel et al., 1999; Schobersberger et al., 1995b), stimulate nitric oxide synthase gene expression (Schobersberger et al., 1995a), and together with cyclic-GMP, induce redox-sensitive proto- oncogene c-fos (Uberall et al., 1994).
Elevated levels of neopterin have been detected in urine (Reibnegger et al., 1984) and plasma (Kremsner et al., 1989; Ringwald et al., 1991) of malaria patients infected with Plasmodium falciparum. When 71 Thai patients with acute, uncomplicated falciparum malaria were treated with quinine and tetracycline for 7 days, the neopterin levels in urine peaked on days 3-5 following the start of treatment before decreasing towards the normal range on days 6-8 (Brown et al., 1990). Similar trends were observed in other studies (Kremsner et al., 1996; Ringwald et al., 1991). Higher concentrations of neopterin as well as IFN-γ were found more frequently in patients infected with malaria for the first time than experienced patients (Brown et al., 1990;
Ringwald et al., 1991) which suggested that repeated malaria infection and antigen exposure results in a progressive suppression of the T-cell-macrophage interaction mediated by IFN-γ. During seven days of quinine antimalarial therapy, serum neopterin levels remained elevated in children who were still found to have persisting anaemia one month after completing treatment;
but the neopterin levels declined significantly in the children who had normal haemoglobin levels a month after completing treatment (Biemba et al., 1998). The elevated neopterin levels suggested the persistence of the Th-1 mediated immune response and associated macrophage activation may be involved in the pathogenesis of the lingering anaemia after the treatment of malaria. Children with severe P. falciparum malaria with respiratory distress, which is a symptom of underlying acidosis, had significantly higher levels of neopterin than those malaria- infected children without respiratory distress (Awandare et al., 2006a). These observations show that monitoring the neopterin levels of a patient during treatment of malaria with antimalarial drugs cannot be used to give an indication if the antimalarial drugs themselves have any effect on macrophage activation and neopterin production.
Lysates of P. falciparum-infected erythrocytes were found to stimulate neopterin secretion from U937 cells after 48 hours co-incubation (Facer, 1995) and this secretion was enhanced by IFN- γ. Several P. falciparum antigens also activated U937 cells to secrete neopterin and produced a similar response when cultured with peripheral blood mononuclear cells for 7 days (Facer, 1995). The malarial pigment, haemozoin, has been shown to have both stimulatory and inhibitory effects on the functions of monocytes and macrophages (Schwarzer et al., 2008), however, the effect of β-haematin, the synthetic equivalent of haemozoin, on neopterin secretion is unknown. In this study, the effects of seven antimalarial drugs (amodiaquine, artemisinin, chloroquine, doxycycline, primaquine, pyrimethamine and quinine), β-haematin, latex beads, non-infected- and P. falciparum-infected-red blood cell lysates on the IFN-γ- induced expression of GTP-cyclohydrolase 1 mRNA were investigated in the human monocytic
cell line, U937. Antibodies were raised against neopterin in chickens and used in the development of a competitive ELISA to detect neopterin in the supernatants of treated U937 cells. Total RNA was isolated from the treated U937 cells and the expression of GTP- cyclohydrolase 1 mRNA was analysed using quantitative RT-PCR and the 2−∆∆CT method.