Influence of cytokines tumor necrosis factor-alpha and interferon-gamma on signaling cascades associated with apoptosis in rat PC12 cells.

During cell-mediated immune response, increased amounts of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) are released. In the present study, we investigated the potential of these two cytokines to mediate apoptosis and to alter signal transduction pathways involved in undifferentiated PC12 cells. To induce apoptosis, the pteridine 7,8-dihydroneopterin (NH2) was used. TNF-alpha alone and TNF-alpha in combination with IFN-gamma led to no alteration in cell viability during 48 h of incubation. TNF-alpha was able to slightly elevate apoptosis compared with cells stimulated with NH2 alone. The combination of TNF-alpha and IFN-gamma almost completely abrogated the rate of apoptosis induced by NH2. Similar degrees of activation of extracellular protein kinase were found after the addition of cytokines or cytokines in combination with NH2. Stress-activated protein kinase (SAPK) was not activated by the cytokines alone, whereas adding the cytokine TNF-alpha to NH2-stimulated cells resulted in activation of SAPK after 15 min.

Induction of apoptosis by 7,8-dihydroneopterin: involvement of radical formation.

Interferon-gamma is a cytokine released in large amounts during cell-mediated immune response. It induces the expression of proinflammatory cytokines and enhances macrophage capacity to secrete reactive oxygen intermediates and the pteridines neopterin and 7,8-dihydroneopterin. To assay the role of these pteridines in the immune system several studies were performed. Thereby, 7,8-dihydroneopterin was found to induce apoptosis in T lymphocytes. In this study we report that caspases are involved in 7,8-dihydroneopterin-mediated apoptosis in Jurkat T cells. In connection with this result we found that 7,8-dihydroneopterin can increase Fas ligand expression detected in Western blot analysis and promoter reporter assays. Antioxidants potently reduced the effect of 7,8-dihydroneopterin on Fas ligand promoter activation suggesting an involvement of oxidative stress. In further investigations, ESR-measurements were performed to evaluate the role of 7,8-dihydroneopterin in the formation of radicals. We found that the pteridine in combination with the spin trap DMPO induces the production of DMPO-OH spin adducts. This reaction was sensitive to the presence of chelated metal ions and could completely be blocked by the addition of superoxide dismutase. These data suggest that 7,8-dihydroneopterin in aqueous solution leads to the formation of .OH radicals via generation of superoxide anion. We hypothesize that an overproduction of radicals caused by high levels of 7,8-dihydroneopterin is likely to be responsible for the pro-apoptotic effects observed in cell cultures and possibly contributes to the pathogenesis of diseases involving immune activation and elevated concentrations of neopterin-derivatives.

Reduced pteridine derivatives induce apoptosis in human neuronal NT2/HNT cells.

Elevated concentrations of the pteridine compound neopterin, usually accompanied by 7,8-dihydroneopterin were found in cerebrospinal fluids of patients with neurodegenerative diseases and central nervous system infections. Here, the potential of pteridines to induce apoptosis of the human neuronal cell line (NT2) was investigated. Reduced neopterin, biopterin- and folate derivatives led to a time-dependent increase of apoptosis of cells. In contrast, non-reduced pteridines did not significantly alter cell survival. After differentiation of neuronal precursor cells to neurons and astrocyte-like cells, similar effects were detected. Antioxidants partly protected NT2 from pteridines-induced apoptosis, suggesting the involvement of reactive oxygen intermediates. In vitro experiments using dichlorofluorescin-diacetate further indicated a direct formation of reactive oxygen species in cells. Results implicate that high concentrations of reduced pteridines, might contribute to the loss of neuronal cells in neurodegenerative diseases.

Synergistic action of protein kinase C theta and calcineurin is sufficient for Fas ligand expression and induction of a crmA-sensitive apoptosis pathway in Jurkat T cells.

Deletion of activated peripheral T cell clones by apoptosis requires the regulated expression of Fas ligand (FasL) and sensitization of these cells to CD95-mediated signaling. To investigate the signaling pathways responsible for FasL expression in T cells, we tested-besides subfamily-selective protein kinase C (PKC) inhibitors - the effect of constitutively active mutants of representatives of all PKC subfamilies, i.e. PKCalpha,epsilon,theta,iota, on FasL luciferase promoter reporter constructs. In synergy with a constitutively active form of protein phosphatase 2B calcineurin (CaN), only PKCtheta, but not PKCalpha,epsilon,iota, preferentially induced FasL promoter reporter activity and, consequently, FasL protein expression in Jurkat T cells. Activation of an inducible PKCtheta AE-estrogen receptor fusion mutant led to a CaN-dependent and rapid FasL reporter activity detected as early as 4 h after addition of 4-hydroxytamoxifen, incidating a direct effect of PKCtheta action on FasL expression. Consistently, in Jurkat T cells, expression of PKCtheta AE / CaN significantly enhanced FasL protein expression and apoptosis in a CD95-dependent manner since cell death was not observed in T cells co-expressing the caspase-8 inhibitor crmA. Taken together, our results support the notion that PKCtheta and CaN are sufficient to regulate apoptosis through FasL expression.

Increased neopterin concentrations in patients with cancer: indicator of oxidative stress?

In vitro, large amounts of neopterin are produced by human monocytes/macrophages upon stimulation with interferon-gamma. In vivo increased neopterin concentrations in human serum and urine indicate activation of cell-mediated (Th1-type) immune response, e.g., during virus infections, autoimmune diseases, allograft rejection and in certain types of malignancy. In various groups of patients with malignant diseases neopterin concentrations correlate to the stage of disease, and higher neopterin concentrations in serum, urine or ascitic fluid were shown to significantly predict worse prognosis regarding relapse and survival. The amounts of neopterin produced by activated monocytes/macrophages correlate with their capacity to release reactive oxygen species (ROS). With this background, neopterin concentrations in body fluids can be regarded as an indirect estimate of the degree of oxidative stress emerging during cell-mediated immune response. Moreover, recently neopterin was found itself to be capable of enhancing toxic effects induced by ROS. In vitro, neopterin derivatives were able to interfere with intracellular signal transduction pathways involved in, e.g., programmed cell death and the induction of proto-oncogene c-fos or nuclear factor-chi B. The data support the view that increased production of ROS--indicated by increased neopterin concentrations--could modulate the development, the proliferation and the survival of malignant cells.

Evidence that atypical protein kinase C-lambda and atypical protein kinase C-zeta participate in Ras-mediated reorganization of the F-actin cytoskeleton.

Expression of transforming Ha-Ras L61 in NIH3T3 cells causes profound morphological alterations which include a disassembly of actin stress fibers. The Ras-induced dissolution of actin stress fibers is blocked by the specific PKC inhibitor GF109203X at concentrations which inhibit the activity of the atypical aPKC isotypes lambda and zeta, whereas lower concentrations of the inhibitor which block conventional and novel PKC isotypes are ineffective. Coexpression of transforming Ha-Ras L61 with kinase-defective, dominant-negative (DN) mutants of aPKC-lambda and aPKC-zeta, as well as antisense constructs encoding RNA-directed against isotype-specific 5' sequences of the corresponding mRNA, abrogates the Ha-Ras-induced reorganization of the actin cytoskeleton. Expression of a kinase-defective, DN mutant of cPKC-alpha was unable to counteract Ras with regard to the dissolution of actin stress fibers. Transfection of cells with constructs encoding constitutively active (CA) mutants of atypical aPKC-lambda and aPKC-zeta lead to a disassembly of stress fibers independent of oncogenic Ha-Ras. Coexpression of (DN) Rac-1 N17 and addition of the phosphatidylinositol 3'-kinase (PI3K) inhibitors wortmannin and LY294002 are in agreement with a tentative model suggesting that, in the signaling pathway from Ha-Ras to the cytoskeleton aPKC-lambda acts upstream of PI3K and Rac-1, whereas aPKC-zeta functions downstream of PI3K and Rac-1. This model is supported by studies demonstrating that cotransfection with plasmids encoding L61Ras and either aPKC-lambda or aPKC-zeta results in a stimulation of the kinase activity of both enzymes. Furthermore, the Ras-mediated activation of PKC-zeta was abrogated by coexpression of DN Rac-1 N17.

7,8-Dihydroneopterin-induced apoptosis in Jurkat T lymphocytes: a comparison with anti-Fas- and hydrogen peroxide-mediated cell death.

Activated cell-mediated immunity, associated for example with HIV infection, is accompanied by elevated concentrations of neopterin and 7,8-dihydroneopterin. Recent data have indicated a role of neopterin derivatives in virus activation and apoptotic cell death, processes likely to involve the action of oxygen free radicals. Because T cell death in AIDS is likely to involve the Fas/Fas ligand system and the action of oxygen free radicals and 7,8-dihydroneopterin, we compared the kinetics and sensitivity of apoptotic cell death of human leukemic Jurkat T cells to that of treatments with 7,8-dihydroneopterin, anti-Fas, and H2O2. Upon incubation with 5 mM 7,8-dihydroneopterin and 50 microM hydrogen peroxide over a period of 24 hr, bimodal kinetics were observed with peaks at 5.5 hr (7,8-dihydroneopterin, 13.1%; H2O2, 11.4%) and at 24 hr (7,8-dihydroneopterin, 11.2%; H2O2, 13.2%). In contrast, anti-Fas (20 ng/mL)-induced apoptosis increased steadily over time, peaking at 11 hr (43.2%). Interestingly, anti-Fas-induced apoptosis was suppressed upon co-incubation with 7,8-dihydroneopterin and H2O2 by 62% and 68%, respectively. We also compared the sensitivity to drug treatments of apoptosis induced by 7,8-dihydroneopterin, anti-Fas antibodies, and H2O2. 7,8-Dihydroneopterin-mediated, and similarly anti-Fas- and H2O2-mediated, apoptosis was not inhibited by a broad range of pharmacological inhibitors, such as actinomycin D, cycloheximide, cyclosporin A, and various protein kinase inhibitors. On the contrary, inhibitors with antioxidant abilities, such as pyrrolidinedithiocarbamate, significantly blocked 7,8-dihydroneopterin-, H2O2- as well as anti-Fas-mediated apoptosis. These results imply that 7,8-dihydroneopterin-, H2O2-, and anti-Fas-mediated cell death might involve related redox sensitive signal transduction pathways.

Neopterin induces nitric oxide-dependent apoptosis in rat vascular smooth muscle cells.

Numerous studies indicate that proinflammatory substances like tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) as well as macrophage-derived neopterin are increased in atherosclerotic tissue and thus are potentially involved in the process of atherogenesis. Since apoptotic death of vascular smooth muscle cells (VSMC) is reported to occur in atherosclerotic lesions, we investigated the effects of neopterin, TNF-alpha, and IFN-gamma on apoptosis in cultured VSMC. Morphological changes characteristic of apoptosis as well as DNA fragmentation were detected in cells treated with neopterin, TNF-alpha/IFN-gamma, and neopterin + TNF-alpha/IFN-gamma. Simultaneously, neopterin, TNF-alpha/IFN-gamma, and neopterin + TNF-alpha/IFN-gamma led to inducible nitric oxide synthase (iNOS) gene expression and nitric oxide (NO) synthesis. NO generation was significantly reduced when cells were cotreated with the competitive iNOS inhibitor aminoguanidine. This was accompanied by decreased percentual apoptosis as detected by FACS analysis using all kinds of stimuli. We conclude that neopterin as well as TNF-alpha/IFN-gamma are potent mediators of apoptotic death in VSMC which is at least in part triggered by NO synthesis induced by these proinflammatory mediators.

Structure and function of HIV-1 and SIV Tat proteins based on carboxy-terminal truncations, chimeric Tat constructs, and NMR modeling.

To further define the structure and function of the domains in HIV-1 and SIV Tat proteins, chimeric Tat cDNA expression constructs were generated with crossover points at the carboxy-terminal end of the cysteine rich domain. The chimera containing the amino-terminal region of SIV and carboxy-terminal region of HIV exhibited activity similar to HIV-1 Tat and SIV Tat on both the HIV-1 and SIV LTRs. In contrast, the reciprocal chimera functioned poorly. As determined by the activity of carboxy-terminal truncation mutants, the region immediately downstream of the basic domain is critical for efficient transactivation by HIV-1 Tat, but not SIV Tat protein. In this report, we present a model for Tat domains based on NMR data and the known functional properties of Tat protein. According to our modeling two sites for protein : protein interactions are present in HIV-1 and SIV Tat proteins. Site I, which is presumably involved in cyclin T binding, is similar in both HIV-1 and SIV Tat proteins as well as in Tat chimeras. Site II, however appears structurally different in HIV-1 and SIV Tat models, although in both cases is comprised of amino and carboxy-terminal residues. Differences in Site II may thus account for the differential activities of HIV-1 and SIV Tat carboxy-terminal truncations. Site II in the poorly active chimera differs significantly from that found in HIV-1 and SIV Tat proteins. The two site structural model presented here may have important implications for the role of Tat in HIV pathogenesis and may provide insights for the design of Tat vaccines and targeted therapeutics.

trans-Activation of the HIV type 1 promoter by 7,8-dihydroneopterin in vitro.

Infection with human immunodeficiency virus type 1 and 2 is associated with elevated concentrations of neopterin, released in large quantities by human macrophages on stimulation with interferon gamma. Evidence has suggested a potential role of neopterin derivatives in oxygen radical-mediated processes. Here we show that the redox-sensitive transcription factors AP-1 and NF-kappaB are activated by 7,8-dihydroneopterin, either directly (AP-1), or by the synergistic action with tumor necrosis factor alpha (NF-kappaB). We could further demonstrate that 7,8-dihydroneopterin enhances HIV-1 expression as shown in transient transfection assays using HIV-1 CAT promoter-reporter gene constructs. In sera of HIV+ patients 7,8-dihydroneopterin significantly correlated with neopterin and HIV-1 p24 antigen. On the basis of our data we therefore assume that 7,8-dihydroneopterin might augment progression to higher stages of HIV-associated disease.

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 microM) as well as 7,8-dihydroneopterin (1-1000 microM) resulted in a significant increase of percent apoptotic cells (measured by FACS analysis). Coincubation of both pteridines with the cytomix (interferon-gamma plus tumor necrosis factor-alpha) 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.

Role of 7,8-dihydroneopterin in T-cell apoptosis and HTLV-1 transcription in vitro.

Adult T-cell leukemia is associated with high levels of neopterin, released in large amounts from human macrophages upon stimulation with interferon-gamma. Recent data suggested a potential role of neopterin-derivatives in oxygen radical-mediated processes, and evidence accumulates that oxidative stress is involved in the pathogenesis of viral diseases. We now report that increased concentrations of 7,8-dihydroneopterin may lead to enhanced apoptosis and disturbance of the redox-balance of human leukemic Jurkat T cells. Additionally, we demonstrate that 7,8-dihydroneopterin and hydrogen peroxide activate the type 1 human T-cell leukemia virus (HTLV-1) long terminal repeat (LTR). Furthermore, we found that the activity of the HTLV-1 transactivator protein Tax is amplified by an elevated concentration of 7,8-dihydroneopterin. Tax did not significantly augment 7,8-dihydroneopterin mediated apoptosis. Based on our data we propose that 7,8-dihydroneopterin may be involved in the progression to higher stages of HTLV-1 associated disease.

Role of neopterin and 7,8-dihydroneopterin in human immunodeficiency virus infection: marker for disease progression and pathogenic link.

Human immunodeficiency virus (HIV) infection is associated with increased concentrations of neopterin derivatives, released in large quantities by human macrophages on stimulation with interferon-gamma (INF-gamma). Neopterin concentrations thus inversely correlate with absolute CD4+ T-cell numbers and strongly predict progression of disease from latency to AIDS. Investigations of hydrogen peroxide-induced chemiluminescence indicated a potential role of neopterin and 7,8-dihydroneopterin in oxygen free radical-mediated processes. Indeed, 7,8-dihydroneopterin is able to enhance tumor necrosis factor alpha (TNF-alpha)-induced apoptosis, accompanied by an increased production of reactive oxygen intermediates (ROIs). In line with this finding, the same combination appears to contribute to the upregulation of HIV replication due to activation of nuclear factor-kappa B (NF-kappa B), a central enhancer element of the HIV LTR promoter. Thus, besides the role of neopterin as sensitive indicator of disease activity in HIV infection, neopterin derivatives apparently are associated with the cascade of events that regulate the HIV production in infected individuals.

Protein kinase C-theta isoenzyme selective stimulation of the transcription factor complex AP-1 in T lymphocytes.

T-lymphocyte stimulation requires activation of several protein kinases, including the major phorbol ester receptor protein kinase C (PKC), ultimately leading to induction of lymphokines, such as interleukin-2 (IL-2). The revelant PKC isoforms which are involved in the activation cascades of nuclear transcription factors involved in IL-2 production have not yet been clearly defined. We have examined the potential role of two representative PKC isoforms in the induction of the IL-2 gene, i.e., PKC-alpha and PKC-theta, the latter being expressed predominantly in hematopoietic cell lines, particularly T cells. Similar to that of PKC-alpha, PKC-theta overexpression in murine EL4 thymoma cells caused a significant increase in phorbol 12-myristate 13-acetate (PMA)-induced transcriptional activation of full-length IL-2-chloramphenicol acetyltransferase (CAT) and NF-AT-CAT but not of NF-IL2A-CAT or NF-kappaB promoter-CAT reporter gene constructs. Importantly, the critical AP-1 enhancer element was differentially modulated by these two distinct PKC isoenzymes, since only PKC-theta but not PKC-alpha overexpression resulted in an approximately 2.8-fold increase in AP-1-collagenase promoter CAT expression in comparison with the vector control. Deletion of the AP-1 enhancer site in the collagenase promoter rendered it unresponsive to PKC-theta. Expression of a constitutively active mutant PKC-theta A148E (but not PKC-alpha A25E) was sufficient to induce activation of AP-1 transcription factor complex in the absence of PMA stimulation. Conversely, a catalytically inactive PKC-theta K409R (but not PKC-alpha K368R) mutant abrogated endogenous PMA-mediated activation of AP-1 transcriptional complex. Dominant negative mutant Ha-RasS17N completely inhibited the PKC-O A148E-induced signal, PKC-O. Expression of a constitutively active mutant PKC-O A148E (but not PKC-alpha A25E) was sufficient to induce activation of AP-1 transcription factor complex in the absence of PMA stimulation. Conversely, a catalytically inactive PKC-O K409R (but not PKC-alpha K368R) mutant abrogated endogenous PMA-mediated activation of AP-1 transcriptional complex. Dominant negative mutant Ha-enRasS17N completely inhibited in the PKC-O A148E-induced signal, identifying PKC-theta as a specific constituent upstream of or parallel to Ras in the signaling cascade leading to AP transcriptional activation.

Effects of neopterin-derivatives on H2O2-induced luminol chemiluminescence: mechanistic aspects.

Neopterin, 6-D-erythro-1',2',3'-trihydroxypropyl-pterin, and its dihydroform, 7,8-dihydro-neopterin, are synthesized by human monocytes/macrophages upon stimulation by interferon-gamma. In the presence of iron chelator complexes neopterin enhances hydrogen peroxide-induced luminol chemiluminescence at neutral or slightly alkaline pH (7.5). In contrast, 7,8-dihydroneopterin scavenges chemiluminescence independently from the pH value and iron. In this study, we explored in more detail the mechanism possibly involved: analysis of the reaction products shows that 7,8-dihydroneopterin is oxidized and degraded to 7,8-dihydroxanthopterin and xanthopterin, whereas the neopterin molecule is not chemically altered during the chemiluminescence reaction. Investigations of the neopterin-induced effect show that mannitol, a scavenger of hydroxyl radicals, does not alter the enhancing effect of neopterin. L-histidine, which scavenges singlet oxygen almost as effective as hydroxyl radicals, reduces the enhancing effect of neopterin. However, singlet oxygen was not detectable during the reaction by measuring monomol light emission (1270 nm). When replacing hydrogen peroxide by 3-morpholinosydnonimine, a generator of hydroxyl radicals, or naphthalene-endoperoxide, a generator of singlet oxygen, in the luminol chemiluminescence assay, neopterin shows no enhancing effect irrespective of the presence of iron-(III)-EDTA. The data suggest that neopterin enhances hydrogen peroxide-induced luminol chemiluminescence in the presence of iron-(III)-EDTA by formation of a catalytic complex that seems to favor the formation of oxygen intermediates which derive from hydrogen peroxide and react with luminol.

7,8-Dihydroneopterin upregulates interferon-gamma promoter in T cells.

Activated cell-mediated immunity is accompanied by elevated concentrations of interferon-gamma leading to the secretion of neopterin-derivatives which are known as sensitive immune activation markers in clinical laboratory diagnosis. Recent data imply a potential role of neopterin-derivatives in oxygen-free radical-mediated processes and a direct impact of 7,8-dihydroneopterin on tumor necrosis factor alpha-mediated programmed cell death. We report here that 7,8-dihydroneopterin and hydrogen peroxide upregulate the production of interferon-gamma, thereby establishing an autocrine feed-back loop. Data put emphasis on the role of neopterin-derivates within the cytokine network.

Soluble Receptors for Tumor Necrosis Factor and Neopterin as Parameters of Cell-Mediated Immune Activation.

Tumor necrosis factor-α is a pleiotropic cytokine which is capable of inducing numerous immunological and biochemical changes in target cells and tissues, Soluble tumor necrosis factor-α receptors function to modulate the biological properties of tumor necrosis factor-α by enhancing or counteracting its action. In various pathologic states the production and release of soluble tumor necrosis factor receptors may mediate host response and determine the course and outcome of disease by the interaction with tumor necrosis factor-α and competing with cell surface receptors. The actual and forthcoming research of soluble tumor necrosis factor receptors in body fluids such as plasma or serum is a new tool to gain information about immune processes and is providing valuable insight into a variety of pathological conditions. In various diseases concentrations of sTNF-Rs correlate strongly with levels of neopterin, large amounts of which being released from human monocytes/macrophages upon stimulation with interferon-γ. Like neopterin, sTNF-Rs levels show high accuracy in the follow-up and prognosis of various diseases and correlate with the clinical stage and the progression of human immunodeficiency virus infection and sepsis. In cancer and autoimmune diseases, measurement of sTNF-Rs and neopterin provides useful information for monitoring. The quantification of sTNF-Rs and neopterin concentrations often provide even superior information than that obtained with classical disease markers, probably due to the direct involvement of the "interferon-γ/tumor necrosis factor-α system" in the pathogenetic mechanisms of the diseases. Data imply that measurement of sTNF-Rs, especially of the 75kD-type, is a useful adjunct for quantification of the Th-1 type immune response, sharing many characteristics with neopterin.

Detection of bacterial pyrogens on the basis of their effects on gamma interferon-mediated formation of neopterin or nitrite in cultured monocyte cell lines.

In a number of mammalian cell types, pteridine biosynthesis from guanosine 5'-triphosphate and formation of nitric oxide from L-arginine are induced by gamma interferon (IFN-gamma) and bacterial lipopolysaccharide (LPS). We assessed the possibility of using such metabolic alterations for the in vitro detection of pyrogens. Products from gram-negative and gram-positive bacteria and related synthetic compounds were tested for their potential to induce either of these pathways. Stimulation of pteridine biosynthesis was monitored as the formation of neopterin in the human myelomonocytic cell line THP-1. The formation of nitric oxide was determined as nitrite in murine J774A.1 macrophage cultures. The substances tested included toxic and detoxified parts of LPS and lipid A from Escherichia coli, Salmonella typhimurium, Salmonella minnesota, and Klebsiella pneumoniae as well as lipoteichoic acid and toxic shock syndrome toxin 1 from Staphylococcus aureus. Furthermore, two cell wall compounds from Mycobacterium tuberculosis, trehalose 6,6'-dimycolate and N-acetylmuramyl-L-alanyl-D-isoglutamine, which are active components of Freund's adjuvant, were used. When applied as a single stimulus, only the whole LPS molecule potently stimulated neopterin or nitrite formation. Lipid A and products from gram-positive bacteria were weakly active. For neopterin formation, lipid A required the presence of fetal calf serum. Besides detoxified LPS and independently from the presence of serum, all bacterial compounds tested strongly increased the effects mediated by IFN-gamma. Our results show that bacterial pyrogens can be detected by monitoring the formation of neopterin or nitrite. This may provide a basis for the development of an in vitro assay for the detection of pyrogenic contamination with the aim of replacing the currently used animal test.