Walter Däubener

Restriction of Toxoplasma gondii Growth in Human Brain Microvascular Endothelial Cells by Activation of Indoleamine 2,3-Dioxygenase

ABSTRACT One of the first steps in the development of cerebral toxoplasmosis is the penetration of the blood-brain barrier, which is comprised of microvascular endothelial cells. We examined the capacity of human brain microvascular endothelial cells (HBMEC) to interact withToxoplasma gondii. We found that stimulation of HBMEC with gamma interferon (IFN-γ) resulted in the induction of toxoplasmostasis. The capacity of HBMEC to restrictToxoplasma growth after IFN-γ stimulation was enhanced in the presence of tumor necrosis factor alpha (TNF-α). In addition, we found that IFN-γ induced a strong induction of indoleamine 2,3-dioxygenase (IDO) activity in HBMEC, and this enzyme activity was enhanced by costimulation with TNF-α. The addition of excess amounts of tryptophan to the HBMEC cultures resulted in a complete abrogation of the IFN-γ–TNF-α-mediated toxoplasmostasis. We therefore conclude that IDO induction contributed to the antiparasitic effector mechanism inducible in HBMEC by IFN-γ and TNF-α.

Ifn-γ Activated Indoleamine 2,3-Dioxygenase Activity in Human Cells is an Antiparasitic and an Antibacterial Effector Mechanism

In nearly all human cells IFN-gamma stimulation leads to an activation of indoleamine 2,3-dioxygenase (IDO) activity, which is responsible for anti-toxoplasma and anti-chlamydia effects. We have recently shown that IDO activation is also a defense mechanism against extracellular beta-hemolytic streptococci groups A, B, C and G in human glioblastoma cells, fibroblasts and macrophages. Similar effects were also seen with enterococci and in approximately 65% of staphylococci tested, including multiresistant strains of both species. In addition, we have found that IDO activity is differentially regulated in different cells. For example we have found that TNF-alpha enhances IFN-gamma induced IDO activity and antimicrobial effect in human glioblastoma cells whereas both IFN-gamma mediated effects were blocked by TNF-alpha as well as by IL-1 in a human uroepithelial cell line. We were able to show that the IL-1 and TNF-alpha mediated inhibition of IFN-gamma-induced IDO activity in uroepithelial cells is due to stimulation of inducible nitric oxide synthase. In human astrocytoma cells, IL-1 and TNF-alpha did not inhibit IDO activity and in concordance with this finding these cells did not show a detectable nitric oxide production.

Nitric Oxide-Mediated Regulation of Gamma Interferon-Induced Bacteriostasis: Inhibition and Degradation of Human Indoleamine 2,3-Dioxygenase

ABSTRACT Tryptophan depletion resulting from indoleamine 2,3-dioxygenase (IDO) activity within the kynurenine pathway is one of the most prominent gamma interferon (IFN-γ)-inducible antimicrobial effector mechanisms in human cells. On the other hand, nitric oxide (NO) produced by the inducible isoform of NO synthase (iNOS) serves a more immunoregulatory role in human cells and thereby interacts with tryptophan depletion in a number of ways. We investigated the effects of NO on IDO gene transcription, protein synthesis, and enzyme activity as well as on IDO-mediated bacteriostasis in the human epithelial cell line RT4. IFN-γ-stimulated RT4 cells were able to inhibit the growth of Staphylococcus aureus in an IDO-mediated fashion, and this bacteriostatic effect was abolished by endogenously produced NO. These findings were supported by experiments which showed that IDO activity in extracts of IFN-γ-stimulated cells is inhibited by the chemical NO donors diethylenetriamine diazeniumdiolate, S-nitroso-l-cysteine, and S-nitroso-N-acetyl-d,l-penicillamine. Furthermore, we found that both endogenous and exogenous NO strongly reduced the level of IDO protein content in RT4 cells. This effect was not due to a decrease in IDO gene transcription or mRNA stability. By using inhibitors of proteasomal proteolytic activity, we showed that NO production led to an accelerated degradation of IDO protein in the proteasome. This is the first report, to our knowledge, that demonstrates that the IDO is degraded by the proteasome and that NO has an effect on IDO protein stability.

A new, simple, bioassay for human IFN-γ

Abstract IFN-γ induces the production of N -formyl-kynurenine from L -tryptophan in various cell types by the induction of the enzyme indoleamine 2,3-dioxygenase (IDO). The IFN-γ induced IDO activity in the glioblastoma cell line 86HG39 and cells of clone 2D9 derived from this cell line was found to be greater than that in Hela cells and U373MG cells. Consequently 2D9 cells were used in all subsequent experiments. The determination of kynurenine in the supernatant of IFN-γ activated cells was performed photometrically using a microplate reader. It was found that the amount of kynurenine produced was directly proportional to the amount of IFN-γ used to activate cells. The detection limit for IFN-γ of this assay was 20 U/ml. The induction of L -tryptophan degradation was specific for IFN-γ since neither IFN-α, IFN-β, IL-1, IL-2, IL-6, GM-CSF nor TNFα induced the production of detectable amounts of kynurenine by 86HG39 and 2D9 cells. Furthermore, a mab directed against IFN-γ was able to completely block the IFN-γ induced IDO activation. This bioassay was used to determine the IFN-γ content of supernatants harvested from toxoplasma antigen specific human T cell lines and clones. This assay gave reproducible results which correlated well with the IFN-γ content detected in the same samples using a commercially available ELISA kit. Furthermore in the case of T cell supernatant stimulated 2D9 cells a mab directed against IFN-γ was able to completely block IDO induction. We conclude that the measurement of kynurenine production induced by IFN-γ can be used to determinate IFN-γ content. This is a simple bioassay which can be performed with standard laboratory equipment.

Interferon-γ-Induced Activation of Indoleamine 2,3-Dioxygenase in Cord Blood Monocyte-Derived Macrophages Inhibits the Growth of Group B Streptococci

Neonatal sepsis is most often caused by group B streptococci (GBS) and is a major cause of death in the neonatal period. The response of the immune system in the newborn child has received much attention and is thought to be deficient in a number of ways. The effector response of neonatal monocyte-derived macrophages (MDM) was investigated. Interferon-gamma induced the activation of indoleamine 2,3-dioxygenase in MDM and inhibited the growth of GBS. Both effects were enhanced by the addition of tumor necrosis factor-alpha to the culture conditions. The coincident supplementation of L-tryptophan with the bacteria abrogated the bacterial growth inhibition, thus confirming the causative role of L-tryptophan depletion. Control of the extracellular as well as intracellular L-tryptophan levels may thus be one of the effector mechanisms with which the immune system defends the host against GBS dissemination and disease.

Role of Indoleamine-2,3-Dioxygenase in Alpha/Beta and Gamma Interferon-Mediated Antiviral Effects against Herpes Simplex Virus Infections

ABSTRACT Gamma interferon (IFN-γ)-mediated indoleamine-2,3-dioxygenase (IDO) activity in human astrocytoma cells and in native astrocytes was found to be responsible for the inhibition of herpes simplex virus replication. The effect is abolished in the presence of excess amounts of l-tryptophan. Both IFN-α and IFN-β restricted herpes simplex virus replication in both cell types, but (in contrast to the results seen with IFN-γ) the addition of an excess amount of l-tryptophan did not inhibit the induced antiviral effect.

Molecular characterization of TgMIC5, a proteolytically processed antigen secreted from the micronemes of Toxoplasma gondii ☆

During invasion of host cells, Toxoplasma gondii discharges the contents of small, apically located secretory organelles called micronemes. Micronemal proteins are known to be necessary for both parasite motility and invasion of host cells. To further define the contents of Toxoplasma micronemes, we used cell fractionation and secretion-modulating drugs to identify six novel, putative micronemal proteins. In this paper we describe preliminary characterization of one of these novel proteins, TgMIC5. Molecular cloning and DNA sequence analysis of the TgMIC5 cDNA and gene revealed that it encodes a previously identified immunodominant antigen called H4. TgMIC5 also possesses a consensus sequence unique to members of the parvulin family of peptidyl-prolyl cis-trans isomerases (PPIases). TgMIC5 is expressed as a preproprotein, which is proteolytically processed to a proprotein by signal peptidase before being further processed to a mature protein of 22 kDa. Using a combination of protein secretion experiments, immunofluorescence and immunoelectron microscopy, we demonstrated that TgMIC2 is stored in the micronemes of T. gondii tachyzoites before it is secreted into the surrounding medium. Based on its homology with parvulin-like PPIases, TgMIC5 may assist in the folding of other micronemal proteins that function in invasion of host cells by T. gondii tachyzoites.

Differentiation driven by granulocyte-macrophage colony-stimulating factor endows microglia with interferon-γ-independent antigen presentation function

The antigen presentation function of microglial cells was analyzed after differentiation in neonatal mouse brain cell cultures supplemented either with macrophage (M) or granulocyte/macrophage (GM) colony-stimulating factor (CSF). The cells separated from concomitant astrocytes in both culture systems turned out to exhibit cytological characteristics of macrophages and bore MAC-1 and F4/80 markers in a similar way. When comparatively tested for accessory cell function, only microglia developed with GM-CSF were able to efficiently induce antigen-directed proliferation of a series of helper T cell lines representing both the TH1 and TH2 subtype. Antigenic T cell activation by this microglia population was performed without prior stimulation and exceeded that of M-CSF-dependently grown microglial cells, even if those had been pretreated with interferon-gamma (IFN-gamma). In contrast to such difference in function, low cell surface expression of MHC class II or intercellular adhesion molecule-1 determinants proved to coincide in both populations. Correlating with the capacity for antigen presentation, expression of membrane-bound interleukin-1 (IL1)--a costimulatory signal for TH2 cells--was augmented significantly in GM-CSF-grown microglia. In parallel, the interaction only of this microglia population with a selected TH1 cell line was accompanied by maximal release of T cell-stimulating factor, a cytokine recently identified as an IL1-analogous second signal for TH1 cells. Thus, a developmental process is suggested which produces a form of microglia specialized in antigen presentation and thereby acting uncoupled from IFN-gamma.

Cytokine Mediated Regulation of Interferon-Gamma-Induced Ido Activation

Stimulation of human monocyte-derived-macrophages (MDM) with interferon gamma induces the L-tryptophan degrading enzyme indoleamine 2,3-dioxygenase (IDO). It has been well documented that the growth of some intra-cellular parasites such as Chlamydia and Toxoplasma in human fibroblasts and glioblastoma cells is inhibited by IDO mediated L-tryptophan depletion. We have recently shown that IDO induction in cord blood MDM is also responsible for the growth inhibition of extra-cellular group B streptococci and thus for the first time shown an anti-bacterial effect of IDO activation. In view of this immunological function we sought to investigate the regulation, and in particular the downregulation of IDO by the immune system. We describe here the effect of cytokines on IDO activation and in particular the inhibitory function of IL-10, TGF beta and IL-4.

Indoleamine 2,3-dioxygenase mediates cell type-specific anti-measles virus activity of gamma interferon.

ABSTRACT Gamma interferon (IFN-γ) has been shown to be increased in sera from patients with acute measles and after vaccination, to exhibit protective functions in brains of patients with subacute sclerosing panencephalitis, and to mediate a noncytolytic clearance of measles virus (MV) from rodent brains. In order to reveal a possible intracellular antiviral activity in the absence of antigen presentation and cytotoxic T cells, we investigated IFN-γ-induced effects on MV replication in various tissue culture cells. While attenuated MV strains are more sensitive to IFN-α/β than are wild-type strains, IFN-γ inhibits the replication of all MV strains in epithelial, endothelial, and astroglial cells, but not in lymphoid and neuronal cell lines. The antiviral activity induced by IFN-γ correlates with the induction of indoleamine 2,3-dioxygenase (IDO), an enzyme of the tryptophan degradation pathway known to mediate antiviral as well as antibacterial and antiparasitic effects. The IFN-γ-induced antiviral activity can be overcome by the addition of excess amounts of l-tryptophan, which indicates a specific role of IDO in the anti-MV activity. Our data suggest that the IFN-γ-induced enzyme IDO plays an important antiviral role in MV infections of epithelial, endothelial, and astroglial cells.