Schroccksnadel Κ. et air. Neopterin to Monitor Clinical Pathologies Involving IFN-γ Production 75 Pteridines Vol. 15, 2004, pp. 75 - 90 Neopterin to Monitor Clinical Pathologies Involving Interferon-γ Production* Katharina Schroecksnadel1, Christian Murr1, Christiana Winkler1·2, Barbara Wirleitner1--, Lothar C. Fuith', Dietmar Fuchs1·2 'Institute for Medical Chemistry and Biochemistry, Medical University of Innsbruck, 12Ludwig Boltzmann Institute of AIDS Research, 'Division of Gynecology and Obstetrics, Hospital Barmherzige Brüder, Eisenstadt ^Dedicated to Prof. Dr. Dr. h. c. Helmut Wächter on the occasion of his 75,h birthday Abstract Upon stimulation with the cytokine interferon-γ human monocytes/macrophages produce neopterin. Accordingly, measurement of neopterin concentrations in body fluids like blood, urine or cerebrospinal fluid provides infor- mation about activation of immune response involving type 1 Τ helper cells. 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 some neurodegenerative and in cardiovascular diseases. Major diagnostic applications of neopterin measurements are monitoring of the immune status of allograft recip- ients, detection of infectious diseases in blood donations and monitoring of therapy in HIV-infected individuals. Neopterin concentrations also provide prognostic information in HIV-infected individuals and in several malig- nant tumor diseases, high neopterin production at the moment of diagnosis is associated with poorer survival expectations. As high neopterin production is associated with increased production of reactive oxygen species and with low serum concentrations of antioxidants like α-tocopherol, neopterin can be regarded as a marker of oxidative stress caused by an activated immune system. Therefore, by neopterin measurements not only the extent of cellular immune activation, but also the extent of tissue damage caused by reactive oxygen specics may be estimated. Key words: neopterin, laboratory diagnosis, immunobiology, cellular immune response, in vitro testing Introduction class of pteridines which biosynthetically derives from guanosine triphosphate (GTP). Unconjugated pteridi- Neopterin, 2-amino-4-hydroxy-6-(D-erythro-l 2', 3'- nes are synthesized de novo by mammals and by other trihydroxypropyl)-pteridine (Fig.l), belongs to the higher animals (1, 2). In contrast to purines, the bio- logical function of most unconjugated pteridines is still unknown, despite their common occurrence in various organisms. The reason for this comparatively limited research partly lies in analytical difficulties. In some cases the problems of isolation and characterization are caused by the low concentrations of pteridines in bio- logical samples; they are generally present only in trace amounts, are photolabile and hardly soluble in aqueous solution (3, 4). In 1979, neopterin determination was demonstrated to detect and further characterize rare inborn error of Figure 1: Neopterin = 2-amino-4-hydroxy-6-(D-ery- metabolism namely atypical phenylketonuria (PKU) thro- 1', 2', 3'-tryhydroxypropyl)-pteridine (5). In this subgroup of PKU, protein phenylalanine Correspondence to: Dr. Dietmar Fuchs, Institute of Medical Chemistry and Biochemistry, Fritz Pregi Str. 3, A-6020 Innsbruck, Austria, Phone: ++43-512-507-3519, Fax: ++43-512-507-2865, e-mail: [email protected] Pteridines/Vol. 15/No. 3 76 Schroecksnadel Κ. et al.: Ncopterin to Monitor Clinical Pathologies Involving IFN-γ Production hydroxylase is not affected, but the biosynthesis of the like fibroblasts or endothelial cells of several species necessary cofactor 5. 6, 7, 8-tetrahydrobiopterin (H4- produce H4-bip and only scarce amounts of neopterin bip) is disturbed. In the same year, Helmut Wächter derivatives are formed. However, due to a relative and coworkers described increased neopterin concen- deficiency of 6-pyruvoyl tetrahydropterin synthase in trations in patients suffering from malignant diseases human and primate monocytes/macrophages, activa- but also in patients with virus infections (6) and there- tion of GTP cyclohydrolase I leads to an accumulation by opened a much broader area for the application of of H2-nep-triphosphate at the expense of H4-bip. H2- neopterin measurements. While searching for a tumor Nep-triphosphate is then converted by phosphatases to marker, this first result appeared rather disappointing neopterin and H2-nep (Fig. 2) (13). The latter products at the first glancc, however, at the end this finding was are detectable in cell culture supernatants of activated a basis for a remarkable variety of applications of human monocytes/macrophages and in body fluids of neopterin determination (7-11 ). which also contributed humans and primates in a rather constant ratio of to a deeper understanding of the complex interrelation- neopterin per H2-nep of about 1:3 (14). From this ship between immune response and pathogenesis of background, human monocytes/macrophages appear diseases. to constitute the most relevant source of neopterin and H2-nep. Interferon-γ (IFN-γ) is the central stimulus for the activation of GTP cyclohydrolase I, and therefore Immunological background of neopterin biosynthe- in human monocytes/macrophages neopterin and H2- sis nep accumulate (15). Besides, also lipopolysaccharide and tumor necrosis factor-α (TNF-α) superinduce GTP-cyclohydrolase I (EC 3.5.4.16), the key IFN-y-mediated neopterin production (16). enzyme of pteridine biosynthesis, cleaves the purin to Upon stimulation with interferons also monocyte - synthesize 7, 8-dihydroneopterin-(H2-nep)-triphos- derived dendritic cells were found to produce phate (Fig.2). This intermediate is converted by neopterin in similar concentrations as macrophages (17). Other cells like human umbilical vein endothelial lnterferon-γ cells (18) or cultured kidney epithelial cells (19), may produce neopterin upon stimulation with IFN-γ, but to a smaller extent than macrophages (19). In addition, V M Guanosirtriphosphate (GTP) neopterin production by epithelial cells is accompa- GTP-Cyclohydrolase I Γ nied by production of H4-bip (20). As in human dis- ^ 7,8-Dihydroneopterintri phosphate eases like, e.g., infections by human immunodeficien- cy virus (HIV), increased neopterin but no or only slight changes of bioplerin production can be found Neopterin 5,6,7,8-Tetvahydrobiopterin 7,8-Dihydroneopterin (21), in case of immune stimulation by IFN-γ neopterin is mainly produced by human macrophages and not by (human macrophages) (other cells) epithelial cells (22). Figure 2: Biosynthesis of neopterin derivatives in In patients several cytokines such as, e.g. inter- human monocytes/macrophages and monoycte- leukin-(IL)-2, which are able to induce IFN-γ -release derived dendritic cells. Synthesis starts from GTP from Τ cells, also provoke neopterin release (23). (guanosin triphosphate) by induction of GTP cyclohy- Similarly, administration of granulocyte/monocyte d rol as e I via interferon-γ. Large amounts of neopterin stimulating factor (GM-CSF) enhances neopterin pro- derivatives are accumulating at the expense of duction in patients (24) probably by increasing the bioplerin derivatives due to a constitutive deficiency number of monocytes/macrophages. of 6-pyruvoyl tetrahydrobiopterin synthase in the As IL-2 and especially IFN-γ are cytokines typical- cells. Other cells like human fibroblasts or endothelial ly produced by Τ helper (Th) cells subtype 1 (25), cells and cells of other species do synthesize biopterin which are promoting immune response mediated by derivatives and only little neopterin becomes cytotoxic Τ cells, increased production of neopterin in detectable. body fluids can be used to monitor activation of cell- mediated immunity. Accordingly, diseases like, e.g., 6-pyruvoyl tctrahydroptenn synthase to form dihydro- virus infections are accompanied by increased biopterin in the biosynthetic pathway of H4-bip. H4- neopterin production, because cell mediated immune Qip is an essential cofactor of several mono-oxygenas- response dominates (6, 7). On the other hand, in situa- es including phenylalanine-, tyrosine- and tryptophan- tions like, e.g., acute bacterial infections, only moder- s-hydroxylase, and the nitric oxide synthases (12). ate neopterin production can be found. In this case. When GTP-cyclohydrolase I is activated, most cells Th2 cell immune response dominates, characterized by Pteridines/Vol. 15/No. 3 Schroecksnadel Κ. et a!.: Neopterin to Monitor Clinical Pathologies Involving IFN-γ Production 77 the formation of IL-4, -5. -6, -9, -10 and -13, which are duction by the activated human macrophages has not supporting humoral immune response (26). A cross- been demonstrated by now, but recently neopterin regulatory influence exists between Thl-type and Th2- derivatives were found to interfere with reactive oxy- type immune responses, down-regulating each other gen species in vitro (36-42). Therefore it seems possi- when activated (Fig. 3). This is evident from in vitro ble that neopterin derivatives influence the effects and cytotoxicity of reactive oxygen species like hydrogen Th1-type cell G Th2-type cell peroxide which are produced by activated macrophages within the oxidative burst reaction. Formation of nitric oxide by stimulated human macrophages seems to be of limited relevance (43), although in in vitro investigations