Tryptophan depletion resulting from indoleamine 2,3-dioxygenase (IDO) activity within the kynurenine pathway is one of the most prominent gamma interferon (IFN-␥)-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-␥-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-␥-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.The indoleamine 2,3-dioxygenase (IDO) catalyzes the first and rate-limiting step in the kynurenine pathway of tryptophan degradation, the conversion of tryptophan to N-formyl-kynurenine. IDO is induced by gamma interferon (IFN-␥) in the course of an inflammatory response in many human cell types, including macrophages, astrocytes, fibroblasts, and epithelial cells. A strong IDO activity leads to a nearly complete depletion of the essential amino acid tryptophan at the site of infection, which results in growth arrest of several tryptophandependent microorganisms. Among these IDO-sensitive microorganisms are eukaryotic pathogens such as Toxoplasma gondii (10, 36) and bacteria such as group B streptococci (28) and enterococci (29). Furthermore, the immunoregulatory role of tryptophan depletion has recently received much attention. Mellor and colleagues found that T cells are unable to proliferate in a tryptophan-depleted environment and that in vivo IDO activity in the mouse placenta protects allogeneic concepti from being rejected by a T-cell-driven mechanism (33). It has been suggested that first-time activation of T cells in the absence of tryptophan may even result in the development of tolerance to the antigen presented (31).The role of nitric oxide (NO) production by the inducible isoform of NO synthase (iNOS) in human cells is controversial. While having a clearly illustrated antimicrobial potential against a variety of pathogens in rodent cells (reviewed in refer...

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 with Toxoplasma gondii. We found that stimulation of HBMEC with gamma interferon (IFN-␥) resulted in the induction of toxoplasmostasis. The capacity of HBMEC to restrict Toxoplasma growth after IFN-␥ stimulation was enhanced in the presence of tumor necrosis factor alpha (TNF-␣). In addition, we found that IFN-␥ induced a strong induction of indoleamine 2,3-dioxygenase (IDO) activity in HBMEC, and this enzyme activity was enhanced by costimulation with TNF-␣. The addition of excess amounts of tryptophan to the HBMEC cultures resulted in a complete abrogation of the IFN-␥-TNF-␣-mediated toxoplasmostasis. We therefore conclude that IDO induction contributed to the antiparasitic effector mechanism inducible in HBMEC by IFN-␥ and TNF-␣.

Natural killer (NK) cells represent a key component of the innate immune system against cancer. Nevertheless, malignant diseases arise in immunocompetent individuals despite tumor immunosurveillance. Hodgkin lymphoma (HL) is characterized by CD30+ tumor cells and a massive infiltration of immune effector cells in affected lymph nodes. The latter obviously fail to eliminate the malignant cell population. Here, we tested for functional NK cell defects in HL and suggest an improvement of NK function by therapeutic means. We demonstrate that peripheral NK cells (pNK) from patients with HL fail to eliminate HL cell lines in ex vivo killing assays. Impaired NK cell function correlated with elevated serum levels of soluble ligands for NK cell receptors NKp30 (BAG6/BAT3) and NKG2D (MICA), factors known to constrict NK cell function. In vitro, NK cell cytotoxicity could be restored by an NKG2D/NKp30-independent bispecific antibody construct (CD30xCD16A). It artificially links the tumor receptor CD30 with the cytotoxicity NK cell receptor CD16A. Moreover, we observed that NK cells from patients treated with this construct were generally activated and displayed a restored cytotoxicity against HL target cells. These data suggest that reversible suppression of NK cell activity contributes to immune evasion in HL and can be antagonized therapeutically.

Cerebral abscess is a rare complication of staphylococcal septicemia in infants associated with high mortality and morbidity. In the pathogenesis of abscess formation, S. aureus, one major causative agent, interacts with endothelial cells of the brain vessels before reaching the central nervous system. This study examined the growth of S. aureus in human brain microvascular endothelial cells (HBMEC) cultures stimulated with cytokines. IFN-gamma inhibited S. aureus replication by the induction of indoleamine 2,3-dioxygenase (IDO) in HBMEC. This activation of IDO in HBMEC could be shown by RT-PCR and by detection of kynurenine in culture supernatants of activated cells. Resupplementation of L-tryptophan abrogated the inhibitory effect of IFN-gamma on the growth of staphylococci, hence confirming the activation of indoleamine 2,3-dioxygenase as being responsible for the induced bacteriostasis. Addition of TNF-alpha enhanced the IFN-gamma mediated antibacterial effects, whereas TNF-alpha alone had no influence on staphylococcal growth. Stimulation of HBMEC with IFN-gamma failed to activate inducible nitric oxide synthase (iNOS) and subsequent production of nitric oxide (NO). Thus, intra- and extracellular depletion of L-tryptophan seems to be an important process in the defense against staphylococcal brain abscesses by means of creating an unfavorable microenvironment.

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