Affinity imaging mass spectrometry (AIMS): high-throughput screening for specific small molecule interactions with frozen tissue sections.

A novel screening system, using affinity imaging mass spectrometry (AIMS), has been developed to identify protein aggregates or organ structures in unfixed human tissue. Frozen tissue sections are positioned on small (millimetre-scale) stainless steel chips and incubated with an extensive library of small molecules. Candidate molecules showing specific affinity for the tissue section are identified by imaging mass spectrometry (IMS). As an example application, we screened over a thousand compounds against Alzheimer's disease (AD) brain tissue and identified several compounds with high affinity for AD brain sections containing tau deposits compared to age-matched controls. It should also be possible to use AIMS to isolate chemical compounds with affinity for tissue structures or components that have been extensively modified by events such as oxidation, phosphorylation, acetylation, aggregation, racemization or truncation, for example, due to aging. It may also be applicable to biomarker screening programs.

Upregulation of placental indoleamine 2,3-dioxygenase by human chorionic gonadotropin.

We tested the hypothesis that hCG can upregulate human trophoblast indoleamine 2, 3-dioxygenase (INDO), which catalyzes the breakdown of tryptophan in villous circulation. The results revealed that it can. Treatment of human trophoblasts with hCG resulted in a time and dose dependent increase in INDO mRNA and protein levels and its enzyme activity. The hCG effect was hormone specific and required the dimer conformation of hCG. The hCG effect required its receptors and was mediated by a cAMP dependent, but protein kinase A independent, mitogen-activated protein kinase 3/1 (MAPK3/1) signaling mechanism. In summary, the present data demonstrate a novel hCG effect on human placental INDO, which probably plays a key role at maternal fetal interface in preventing fetal rejection.

Indoleamine 2,3-dioxygenase is a novel prognostic indicator for endometrial cancer.

Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-catabolising enzyme inducing immune tolerance. The present study aimed to investigate IDO expression and its prognostic significance in endometrial cancer. Indoleamine 2,3-dioxygenase expression in endometrial cancer tissues (n = 80) was immunohistochemically scored as four groups (IDO-, 1+, 2+, and 3+). The high IDO expression (IDO2+ or 3+) in tumour cells was found in 37 (46.3%) of the 80 cases, and was positively correlated with surgical stage, myometrial invasion, lymph-vascular space involvement, and lymph node metastasis, but not with the histological grade. Patients with high IDO expression had significantly impaired overall survival and progression-free survival (PFS) (P = 0.002 and P = 0.001, respectively) compared to patients with no or weak expression of IDO (IDO- or 1+). The 5-year PFS for IDO-/1+, 2+, and 3+ were 97.7, 72.9, and 36.4%, respectively. Even in patients with early-stage disease (International Federation of Gynecology and Obstetrics I/II, n = 64), the PFS for IDO2+/3+ was significantly poor (P = 0.001) compared to that for IDO-/1+. On multivariate analysis, IDO expression was an independent prognostic factor for PFS (P = 0.020). These results indicated that the high IDO expression was involved in the progression of endometrial cancer and correlated with the impaired clinical outcome, suggesting that IDO is a novel and reliable prognostic indicator for endometrial cancer.

Indoleamine 2,3 dioxygenase and quinolinic acid immunoreactivity in Alzheimer's disease hippocampus.

The present immunohistochemical study provides evidence that the kynurenine pathway is up-regulated in Alzheimer's disease (AD) brain, leading to increases in the excitotoxin quinolinic acid (QUIN). We show that the regulatory enzyme of the pathway leading to QUIN synthesis, indoleamine 2,3 dioxygenase (IDO) is abundant in AD compared with controls. In AD hippocampus, both IDO- and QUIN-immunoreactivity (-IR) was detected in cortical microglia, astrocytes and neurones, with microglial and astrocytic expression of IDO and QUIN highest in the perimeter of senile plaques. QUIN-IR was present in granular deposits within the neuronal soma of AD cortex and was also seen uniformly labelling neurofibrillary tangles. Our data imply that QUIN may be involved in the complex and multifactorial cascade leading to neuro-degeneration in AD. These results may open a new therapeutic door for AD patients.

Role of indoleamine-2,3-dioxygenase in alpha/beta and gamma interferon-mediated antiviral effects against herpes simplex virus infections.

Gamma interferon (IFN-gamma)-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-alpha and IFN-beta restricted herpes simplex virus replication in both cell types, but (in contrast to the results seen with IFN-gamma) the addition of an excess amount of L-tryptophan did not inhibit the induced antiviral effect.

Localization of indoleamine 2,3-dioxygenase in human female reproductive organs and the placenta.

Indoleamine 2,3-dioxygenase (IDO) has been implicated in regulation of feto-maternal tolerance and protection against intracellular and extracellular pathogens. We have studied the expression of IDO in the human female reproductive tract and the placenta by immunohistochemistry. Endometrial glandular and surface epithelial cells showed increasing IDO expression during the course of the menstrual cycle. In term placenta, IDO was irregularly localized to the mesenchymal core and found in isolated areas of the syncytiotrophoblast. In first trimester pregnancy, IDO was not present in placental villi, but was present in glandular epithelium of the decidua, and there were distinctly positive cells scattered in the connective tissue, sometimes in conjunction with lymphoid aggregates. The endothelium of spiral arteries and of capillaries showed some, albeit no generalized, reactivity. IDO was also present in the epithelium of cervical glands and of Fallopian tubes. Specificity of antibody binding was confirmed by Western blot analysis. IDO mRNA was detected in first trimester decidua as determined by RT-PCR. IDO is secreted, as determined by analysis of cervical mucus by high pressure liquid chromatography for the presence of the tryptophan metabolite L-kynurenine, indicating IDO activity. Our results support the concept of IDO providing a mechanism of innate immunity protecting against ascending infections in the female reproductive tract.

Lipopolysaccharide induction of indoleamine 2,3-dioxygenase is mediated dominantly by an IFN-gamma-independent mechanism.

Indoleamine 2,3-dioxygenase (IDO) is a rate-limiting enzyme in the L-tryptophan-kynurenine pathway, which converts an essential amino acid, L-tryptophan, to N-formylkynurenine. It has been speculated that IFN-gamma is a dominant IDO inducer in vivo. The present study used IFN-gamma or TNF-alpha gene-disrupted mice and IFN-gamma antibody-treated mice to demonstrate that lipopolysaccharide (LPS)-induced systemic IDO is largely dependent on TNF-alpha rather than IFN-gamma. IFN-gamma-independent IDO induction was also demonstrated in vitro with LPS-stimulated monocytic THP-1 cells. These findings clearly indicate that there is an IFN-gamma-independent mechanism of IDO induction in addition to the IFN-gamma-dependent mechanism.

Expression of indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase in early concepti.

Indoleamine 2,3-dioxygenase (IDO)-initiated tryptophan degradation in the placenta has been implicated in the prevention of the allogeneic fetus rejection [Munn, Zhou, Attwood, Bondarev, Conway, Marshall, Brown, and Mellor (1998) Science 281, 1191-1193]. To determine how IDO is associated with the development of the fetus and placenta, the time course of IDO expression (tryptophan-degrading activity, IDO protein and IDO mRNA) in the embryonic and extra-embryonic tissues as well as maternal tissues of mice was examined. A high tryptophan-degrading activity was detected in early concepti on days 6.5 and 7.5, whereas IDO protein and its mRNA were not expressed during early gestation, but appeared 2-3 days later, lasted for about 3 days and declined rapidly thereafter. The expression of IDO basically coincided with the formation of the placenta. On the contrary, the early tryptophan-degrading activity was due to gene expression of tryptophan 2,3-dioxygenase (TDO), as shown by Northern and Western analysis. These findings indicate that IDO is transiently expressed in the placenta but that the expression does not last until birth, and that the IDO expression is preceded by expression of another tryptophan-degrading enzyme, TDO, in the maternal and/or embryonic tissues in early concepti.

Indoleamine 2,3-dioxygenase in the human lens, the first enzyme in the synthesis of UV filters.

Tryptophan-derived UV filters have recently been shown to bind to human lens proteins. These UV filter adducts increase in amount with age and appear to be mainly responsible for the yellowing of the lens in man. On the basis of research performed in other tissues, it has been assumed that indoleamine 2,3-dioxygenase (IDO) may be the first and probably rate-limiting enzyme in UV filter biosynthesis. In this study, 25 human lenses were examined by a reliable and sensitive assay method with a monoclonal antibody specific for IDO. IDO activity was detected in all lenses ranging from 26 to 80 years, and there was no clear relationship of IDO activity with age. The mean activity was 0.85 +/- 0.49 nmol of kynurenine formed hr(-1)per lens. IDO expression was found to be localized in the anterior cortex of the lens with little or no activity in the posterior cortex or nucleus. The level in the iris/ciliary body was negligible (<0.05 nmol of kynurenine formed hr(-1)). The lens IDO activity is consistent with UV filter turnover values obtained previously. These findings indicate that IDO is the first enzyme in the UV filter pathway and that UV filter biosynthesis is active even in aged lenses. Yellowing of the aged lens may therefore be preventable by drug-induced suppression of lens IDO activity.

Induction of indoleamine 2,3-dioxygenase in primary human macrophages by HIV-1.

Increased kynurenine pathway metabolism has been implicated in the aetiology of the AIDS dementia complex (ADC). The rate limiting enzyme for this pathway is indoleamine 2,3-dioxygenase (IDO). We tested the efficacy of different strains of HIV-1 (HIV1-BaL, HIV1-JRFL and HIV1-631) to induce IDO in cultured human monocyte-derived macrophages (MDM). A significant increase in both IDO protein and kynurenine synthesis was observed after 48 h in MDM infected with the brain derived HIV-1 isolates, laboratory adapted (LA) HIV1-JRFL, and primary isolate HIV1-631. In contrast, almost no kynurenine production or IDO protein was evident in MDM infected with the high replicating macrophage tropic LA strain, HIV1-BaL. The induction of IDO and kynurenine synthesis by HIV1-JRFL and HIV1-631 declined to baseline levels by day-8 post-infection. Together, these results indicate that only selected strains of HIV-1 are capable of inducing IDO synthesis and subsequent oxidative tryptophan catabolism in MDM.

Tissue distribution of indoleamine 2,3-dioxygenase in normal and malaria-infected tissue.

An immunohistochemical method was developed, using a polyclonal antibody, to detect the enzyme indoleamine 2,3-dioxygenase (IDO) in normal and malaria-infected tissue. Plasmodium berghei ANKA, a cerebral malaria (CM) model, and P. berghei K173, a non-cerebral malaria (NCM) model, were used. It was found that vascular endothelial cells were the primary site of IDO expression in both models of malaria infection and that this response was systemic, with the vascular endothelium of brain, heart, lung, spleen and uterus all staining positive. These results suggest that IDO is part of a systemic host response to parasite infection. Although high levels of IDO production alone may not cause pathology, it is possible that when its production is combined with other features of CM, such as breakdown of the blood-brain barrier (BBB), metabolites of the kynurenine pathway may be able to influence the otherwise tightly regulated, immunologically privileged site of the CNS and cause some of the symptoms and pathology observed.

Expression and purification of recombinant human indoleamine 2, 3-dioxygenase.

Indoleamine 2,3-dioxygenase, the first and rate-limiting enzyme in human tryptophan metabolism, has been implicated in the pathogenesis of many diseases. The human enzyme was expressed in Escherichia coli EC538 (pREP4) as a fusion protein to a hexahistidyl tag and purified to homogeneity in terms of electrophoretic and mass spectroscopic analysis, by a combination of phosphocellulose and nickel-agarose affinity chromatography. The yield of the fusion protein was 1.4 mg per liter of bacterial culture with an overall recovery of 56% from the crude extract. When the culture medium was supplemented with 7 microM hemin, the purified protein contained 0.8 mol of heme per mole of enzyme and exhibited an absorption spectrum consistent with the ferric form of hemoprotein. The pI value of the recombinant enzyme was 7.09 compared with 6.9 for the native enzyme. This was as expected from the addition of the hexahistidyl tag. Similar to the native enzyme, the recombinant enzyme required methylene blue and ascorbic acid for enzyme activity and oxidized not only l-tryptophan but also d-tryptophan and 5-hydroxy-l-tryptophan. The molecular activities for these substrates and their K(m) values were similar to those of the native enzyme, indicating that the addition of the hexahistidyl tag did not significantly affect catalytic activity. The recombinant protein can therefore be used to investigate properties of the native enzyme. This will aid the development of specific inhibitors of indoleamine 2,3-dioxygenase, which may be effective in halting disease progression.

Leukocyte integrin-dependent and antibody-independent cytotoxicity of macrophage against allografts.

Macrophages (Mphis), but not T cells, infiltrating into the rejection site of either i.p. allografted Meth A (H-2d) fibrosarcoma cells in C57BL/6 (B6) (H-2b) mice or BALB/c (H-2d) skin onto B6 mice are cytotoxic against allografts with H-2d specificity. To determine the mechanisms of specific killing of allografts by allograft-induced Mphi (AIM), we raised approximately 5,000 rat monoclonal antibodies (mAbs) against AIM and selected three of them (R1-73, R2-40 and R1-34), each of which inhibited cytotoxic activity against allografts in a dose-dependent manner. The antigens recognized by R1-73, R2-40 and R1-34 mAbs were defined by immunoprecipitation and Western blot analyses as CD11a, CD18 and CD11b, respectively; and the allografts expressed CD54, a ligand of CD11a or CD11b, suggesting leukocyte integrin-dependent killing. Although Ab-dependent cellular cytotoxicity has been recognized as a mechanism of specific killing by Mphis, the infiltration of AIM into the rejection site of allografts far (approximately 6 days) preceded the appearance of serum IgG Ab specific for the allograft. AIM exhibiting full cytotoxic activity against allografts was also induced in the transplantation site of Fcgamma receptor knockout [(B6x129) F1] mice as well as B10.D2 (H-2 compatible with allograft) and B6-xid (X-linked immunodeficiency with B cell-specific defect) strains of mice. In the latter two strains of mice, the levels of serum IgG Ab to the allograft were negligible. Moreover, the cytotoxic activity of AIM against allografts was not affected by pretreatment of the cells with anti-mouse IgG serum, suggesting Ab-independent cytotoxicity.

Induction of indolamine 2,3-dioxygenase in primary human macrophages by human immunodeficiency virus type 1 is strain dependent.

Increased kynurenine pathway metabolism has been implicated in the etiology of AIDS dementia complex (ADC). The rate-limiting enzyme for this pathway is indolamine 2,3-dioxygenase (IDO). We tested the efficacy of different strains of human immunodeficiency virus type 1 (HIV1-BaL, HIV1-JRFL, and HIV1-631) to induce IDO in cultured human monocyte-derived macrophages (MDM). A significant increase in both IDO protein and kynurenine synthesis was observed after 48 h in MDM infected with the brain-derived HIV-1 isolates, laboratory-adapted (LA) HIV1-JRFL, and primary isolate HIV1-631. In contrast, almost no kynurenine production or IDO protein was evident in MDM infected with the highly replicating macrophage-tropic LA strain HIV1-BaL. The induction of IDO and kynurenine synthesis by HIV1-JRFL and HIV1-631 declined to baseline levels by day 8 postinfection. Abundant HIV-1 replication did not reduce the ability of exogenous gamma interferon (IFN-gamma) to induce IDO and kynurenine synthesis in HIV-infected MDM. The addition of anti-IFN-gamma antibody to MDM infected with HIV1-JRFL resulted in an absence of detectable IDO protein after 48 h and a decrease of 64% +/- 1% in supernatant kynurenine concentration. Together, these results indicate that only selected strains of HIV-1 are capable of inducing IDO synthesis and subsequent kynurenine metabolism in MDM. The induction of IDO, while apparently independent of replication capacity, appears to be mediated by a transient production of IFN-gamma in MDM responding to the initial infection with selected strains of HIV-1.

Inhibition of indoleamine 2,3-dioxygenase in human macrophages inhibits interferon-gamma-induced bacteriostasis but does not abrogate toxoplasmastasis.

Induction of indoleamine 2,3-dioxygenase (IDO) by IFN-gamma results in growth inhibition of Toxoplasma and Chlamydia spp. as well as tumor cells. This is caused by the degradation, and therefore depletion, of L-tryptophan necessary for cell protein synthesis. Human macrophages stimulated with IFN-gamma express IDO and inhibit the growth of intracellular toxoplasma and chlamydia as well as that of extracellular bacteria such as group B streptococci. Here we describe experiments in which the L-tryptophan analog, 6-chloro-DL-tryptophan (CDLT) caused a dose-dependent inhibition in the IFN-gamma-induced IDO-mediated L-tryptophan degradation in monocyte-derived macrophages and glioblastoma cells. An inhibition of IDO activity of up to 80 % was observed at concentrations of CDLT of 750 microM. Expression of IDO at this concentration, as shown by Northern blot analysis, was unimpaired. This inhibition of IDO was coupled in glioblastoma cells by a complete abrogation of the IFN-gamma-induced toxoplasmastasis in these cells. IDO inhibition by CDLT in human macrophages resulted in a complete abrogation of the IFN-gamma-induced growth inhibition of streptococci and staphylococci. In contrast to this, IFN-gamma-induced toxoplasmastasis was not inhibited in human macrophages by CDLT-mediated IDO inhibition.

Regulation of indoleamine 2,3-dioxygenase, the first enzyme in UV filter biosynthesis in the human lens. Relevance for senile nuclear cataract.

3-Hydroxykynurenine (3OHKyn), the precursor of UV filters in human lens, is highly autooxidizable, generates H2O2, and binds to lens proteins, yielding a tanned/yellow product resembling senile nuclear cataractous materials. Thus, if 3OHkyn can be shown to be the causative agent in cataract, it may be possible to prevent the disease by lowering the level of 3OHKyn. To this end, indoleamine 2,3-dioxygenase, the first enzyme in UV filter synthesis, was studied using lens epithelial cell lines. The results indicated that the IDO expression is mediated by IFN-gamma. Immuno-suppressants which inhibit production of IFN-gamma may act as anti-cataract agents. Another way to lower the level of 3OHKyn is to use specific inhibitors for IDO. A recombinant human IDO was expressed to develop the inhibitors.

Interferon-gamma-dependent/independent expression of indoleamine 2,3-dioxygenase. Studies with interferon-gamma-knockout mice.

The role of IFN-gamma in the expression of indoleamine 2,3-dioxygenase (IDO), a tryptophan oxidizing enzyme, in mouse tissues under physiological and pathological conditions was investigated using IFN-gamma-knockout mice. The results revealed that i) the expression of IDO in the large intestine or in the cecum is mediated by IFN-gamma, ii) for the systemic IDO induction under endotoxin shock, IFN-gamma is a dominant inducer but not essential, and an IFN-gamma-independent mechanism is also operative, iii) the systemic induction of IDO caused by IL-12 or Pokeweed mitogen is mediated by IFN-gamma, and iv) the constitutive IDO expression in the epididymis is IFN-gamma-independent.

Multiple expression of Ly-6C and accumulation of a Ly-6C pre-mRNA in activated macrophages involved in rejection of an allografted tumor.

We established a monoclonal antibody against a surface antigen of activated macrophages (Møs) involved in the rejection of an allografted mouse tumor, Meth A. Bacteria-elicited Møs also expressed the antigen but resident or inflammatory Møs, or other leukocytes, did not. The antigen was identified as Ly-6C by isolation of cDNA clones encoding it. Two-dimensional immunoblotting revealed that Ly-6C of the Møs exists in multiple forms with a similar size but with different isoelectric points. A Ly-6C pre-mRNA also accumulated abundantly in the Møs. Thus, the multiple expression of Ly-6C and the accumulation of the pre-mRNA are the features of allograft- and bacteria-elicited Møs.

Interferon-gamma-dependent expression of inducible nitric oxide synthase, interleukin-12, and interferon-gamma-inducing factor in macrophages elicited by allografted tumor cells.

We have examined the mechanisms of activation of macrophages (Møs) induced by i.p. allografted Meth A tumor cells (Meth A-Møs) during the rejection of the cells by C57BL/6 mice. Inducible nitric oxide (NO) synthase (iNOS), interleukin-12 (IL-12), and interferon-gamma (IFN-gamma)-inducing factor (IGIF) were transiently expressed in Meth A-Møs during the rejection. The expression was impaired in mice in which the gene encoding IFN-gamma had been disrupted (IFN-gamma-/-). In vitro studies showed that Meth A-Møs from IFN-gamma +/+ mice induced an apoptotic type of cell death in P815 cells, without cell-to-cell contact, in an NO-dependent manner, whereas Meth A-Møs from IFN-gamma-/- mice could not lyse these cells. The iNOS, IL-12, and IGIF expression was also impaired in bacteria-activated Møs from IFN-gamma-/-mice, indicating that IFN-gamma, but not IGIF, would be the initial signal that leads to the activation of Møs in vivo.

Induction in interferon-alpha/beta-treated hepatocytes of the inhibitor of the multiplication of IFN-alpha/beta-resistant Friend leukemia cells.

We reported previously that interferon-alpha/beta (IFN-alpha/beta)-treated hepatocytes in culture released a soluble factor(s) that suppressed the multiplication of an INF-alpha/beta-resistant clone of Friend leukemia cells (FLCs). To characterize the factor(s) further, we first examined the possibility that products of nonparenchymal cells (NPCs) included in small number in the hepatocyte cultures were involved in the inhibitory activity. We prepared cultures of purified adherent NPCs, mostly Kupffer cells, and sinusoidal endothelial cells, and culture supernatants of NPCs pretreated with IFN-alpha/beta were tested for the inhibitory activity for FLC multiplication. IFN did not induce any inhibitory activity in NPC cultures, whereas LPS-stimulated NPCs cultivated in parallel released several inhibitory factors including tumor necrosis factor-alpha (TNF-alpha). To explore the possibility that IFN augmented the release of hepatocyte cytosolic proteins, including arginase, we compared the inhibitory activity in culture supernatant of IFN-treated hepatocytes with that found in hepatocyte extract by anion-exchange chromatography. The IFN-induced inhibitory activity was eluted at relatively high salt concentration as a single peak, while the inhibitory activity in hepatocyte extract was co-eluted with arginase at low salt concentration. These results suggested that IFN induced production by hepatocytes of an inhibitor of FLC multiplication.