Interferon-induced indoleamine 2,3-dioxygenase activity in human mononuclear phagocytes.

Interferon (IFN)-induced tryptophan degradation, catalyzed by indoleamine 2,3-dioxygenase (IDO), has been shown to mediate antimicrobial activity in epithelial cells. IDO activity has also been augmented in peripheral blood mononuclear cells (PBMC) treated with IFN or interleukin-2 (IL-2). The effector cells in this population have now been further characterized. PBMCs were isolated from normal donors, separated into monocyte and lymphocyte populations by plastic adherence, treated with IFN or IL-2, and cultivated in medium supplemented with [3H]tryptophan. Culture supernatants were collected after a 48-h incubation and fractionated by high-performance liquid chromatography; radioactivity was determined in fractions corresponding to tryptophan and its metabolites. IFN-gamma and IFN-beta induced IDO activity only in monocytes (plastic-adherent, nonspecific esterase-positive PBMCs). The induction of IDO activity by IL-2 required both monocytes and lymphocytes. Interaction was required between these populations for induction of IDO by IL-2, due to production of IFN-gamma by T lymphocytes, with subsequent IFN-gamma-mediated induction of IDO in monocytes. A number of myeloid cell lines as well as monocyte-derived macrophages were also tested for their ability to be induced to degrade tryptophan in response to IFN treatment. Monocyte-derived macrophages were found to retain their capacity to be induced by IFN-gamma and IFN-beta to degrade tryptophan after differentiation, and to possess seven times more IDO activity per cell than IFN-induced monocytes. However, the presence of lipopolysaccharide (LPS) in the culture medium was required for the maximum induction of IDO activity by IFN-beta. Furthermore, higher concentrations of LPS were sufficient to induce IDO activity in macrophages in the absence of exogenous IFN.

Potentiation of interferon-induced indoleamine 2,3-dioxygenase mRNA in human mononuclear phagocytes by lipopolysaccharide and interleukin-1.

Previous studies have shown that interleukin-1 (IL-1) enhances interferon (IFN)-gamma-induced indoleamine 2,3-dioxygenase (IDO) enzymatic activity in human monocyte-derived macrophages by increasing expression of IDO mRNA. The objectives of this study were to see if IL-1 also enhances IFN-beta-induced IDO activity by increasing specific mRNA expression and to determine if lipopolysaccharide (LPS) enhances IFN-induced IDO activity in a similar manner. Macrophages were treated with combinations of IFN-beta or IFN-gamma as inducer and LPS or IL-1 as potentiator. After 48 h, IDO mRNA expression was assessed by RT-PCR, and IDO activity was determined by HPLC. LPS alone induced IDO mRNA expression and also increased IDO mRNA expression induced by either type of IFN. Furthermore, IL-1 enhanced IFN-beta-induced IDO mRNA expression. When IDO mRNA was assessed 6 h after treatment, mRNA was detected at concentrations of IFNs or potentiator or both in which enzymatic activity at 48 h was undetectable. Thus, although the mechanism of potentiation of IFN-induced IDO by LPS and by IL-1 involves increased expression of IDO mRNA, it appears that temporal differences in IDO mRNA expression are also important.

Interleukin-1 enhances indoleamine 2,3-dioxygenase activity by increasing specific mRNA expression in human mononuclear phagocytes.

The objective of this study was to determine the utility of the THP-1 monocytic leukemia cell line as a model for analyzing molecular mechanisms involved in enhancement of interferon (IFN)-gamma-induced indoleamine dioxygenase (IDO) activity by interleukin-1 (IL-1). Following treatment of THP-1 cells with combinations of IFN-gamma and IL-1, IDO activity and IDO mRNA were quantified by HPLC and radioanalytic imaging of RT-PCR products, respectively. IL-1 increased the amount of IDO activity and the expression of IDO mRNA in IFN-treated cells; IL-1 alone had no effect on untreated THP-1 cells. Because IDO gene regulation might differ between immature THP-1 cells and mature macrophages, experiments were repeated using primary macrophage cultures. IFN-gamma induced IDO activity, and IDO mRNA was expressed in a dose-dependent manner. In the presence of IL-1, 10 times less IFN was required to obtain the same amount of IDO mRNA and IDO activity. Furthermore, IL-1 alone increased IDO mRNA expression. It appears that unlike what was observed in THP-1 cells, IL-1 transcriptionally activates the IDO gene in primary macrophages. However, increases in IDO activity were not observed following treatment with IL-1 alone. Although the THP-1 cell may be used to model cytokine potentiation of IFN-induced IDO activity, some differences in regulation between THP-1 cells and primary macrophage cultures may exist.

Tumor necrosis factor-alpha and lipopolysaccharide enhance interferon-induced antichlamydial indoleamine dioxygenase activity independently.

In macrophages, interleukin-1 (IL-1) and lipopolysaccharide (LPS) enhance the antichlamydial effect of interferon-gamma (IFN-gamma) by increasing indoleamine 2,3-dioxygenase (IDO) activity in a dose-dependent manner. Our objectives were to characterize the antichlamydial effect of tumor necrosis factor-alpha (TNF-alpha) on IFN-induced IDO activity and to establish the relationship between LPS and TNF-alpha in IDO potentiation. TNF-alpha inhibited chlamydial growth in a dose-dependent manner only in IFN-treated macrophages. Furthermore, excess tryptophan reversed the effect of combined cytokine treatment, indicating that IDO alone was responsible for chlamydial inhibition. The promonocyte THP-1 cell line, previously used to model the effect of IL-1 on IDO mRNA expression, was treated with IFN-gamma and increasing concentrations of LPS or TNF-alpha. IDO mRNA was quantified by RT-PCR, and IDO activity was measured by HPLC at 24 and 48 h after treatment, respectively. Both LPS and TNF-alpha enhanced IDO activity and IDO mRNA expression, with maximal IDO induction at 100 ng/ml LPS or 5 ng/ml TNF-alpha. Anti-TNF-alpha failed to neutralize the effects of LPS treatment, and insufficient TNF-alpha or IL-1 was produced by LPS-treated THP-1 cells to account for the enhancing effect of LPS, indicating that the effect of LPS on IDO was independent of TNF-alpha and IL-1.

Clinical drug-resistant falciparum malaria acquired from cultured parasites.

This is the first reported infection with Plasmodium falciparum acquired from continuous cultures of these parasites. The strain involved, FCR3, was originally isolated from The Gambia, West Africa, and was initially sensitive to chloroquine in vitro. After nearly 4 years of continuous culture without chloroquine pressure, it became resistant to chloroquine in vitro and in vivo, as demonstrated by the failure of both prophylactic and therapeutic regimens to control an accidental inoculation of parasite-infected erythrocytes.

Comparison of inducers of crisis forms in Plasmodium falciparum in vitro.

A variety of known or suspected inducers of crisis form parasites in cultivated Plasmodium falciparum were examined. Sera from Sudanese residents of malaria-endemic areas, sera from American tuberculosis patients, and rabbit sera containing tumor necrosis factor were assayed in vitro for cytotoxic activities against P. falciparum and mouse L-M cell cultures. Inhibition was determined by measurement of incorporation of radiolabeled nucleic acid precursors. When compared to normal serum, parasites grown in the presence of a 1:4 dilution of rabbit sera containing tumor necrosis factor, TB patient sera, or Sudanese sera were metabolically inhibited 73%, 75%, and 95%, respectively. However, only the rabbit sera containing tumor necrosis factor were cytotoxic to L-M cells, inhibiting radiolabel incorporation by 80% at a 1:1,000 serum dilution. These findings suggest that tumor necrosis factor is apparently not responsible for the induction of parasite crisis forms by the inhibitory human sera tested. In addition, human gamma-interferon had no effect on parasite growth.

Stage- and time-dependent effects of crisis form factor on Plasmodium falciparum in vitro.

To determine the stage- and time-dependent effects of crisis form factor (CFF) on Plasmodium falciparum metabolism in vitro, the parasite erythrocytic cycle was divided into sequential 8 hr time intervals, and highly synchronous parasites were exposed to CFF for various lengths of time. Hypoxanthine and phenylalanine incorporation into parasite nucleic acids and proteins, respectively, and glucose consumption by the parasites were compared in cultures grown in CFF-containing or nonimmune sera. The most profound derangement of metabolism occurred in parasites 0-8 hr post-invasion. Inhibition correspondingly decreased in tests started with progressively older parasites. Cultivation in CFF serum for 8 hr caused maximal inhibition of purine and amino acid incorporation; longer periods of exposure did not increase inhibition. In contrast, CFF's effect on glucose consumption varied inversely to the duration of exposure to CFF. As the parasites matured in the presence of CFF, inhibition of the rate of glucose utilization decreased, with little or no reduction in consumption observed as parasites entered schizogony. Of the 3 metabolic parameters studied, hypoxanthine was the most sensitive indicator of metabolic inhibition throughout the cycle.

Medical records documentation of constipation preceding Parkinson disease: A case-control study.

OBJECTIVE: Parkinson disease (PD) may affect the autonomic nervous system and may cause constipation; however, few studies have explored constipation preceding the motor onset of PD. We investigated constipation preceding PD using a case-control study design in a population-based sample. METHODS: Using the medical records-linkage system of the Rochester Epidemiology Project, we identified 196 subjects who developed PD in Olmsted County, MN, from 1976 through 1995. Each incident case was matched by age (+/-1 year) and sex to a general population control. We reviewed the complete medical records of cases and controls in the medical records-linkage system to ascertain the occurrence of constipation preceding the onset of PD (or index year). RESULTS: Constipation preceding PD or the index year was more common in cases than in controls (odds ratio [OR] 2.48; 95% confidence interval [CI] 1.49 to 4.11; p = 0.0005). This association remained significant after adjusting for smoking and coffee consumption (ever vs never), and after excluding constipation possibly induced by drugs. In addition, the association remained significant in analyses restricted to constipation documented 20 or more years before the onset of motor symptoms of PD. Although the association was stronger in women than in men and in patients with PD with rest tremor compared with patients with PD without rest tremor, these differences were not significant. CONCLUSIONS: Our findings suggest that constipation occurring as early as 20 or more years before the onset of motor symptoms is associated with an increased risk of Parkinson disease.

Anemia or low hemoglobin levels preceding Parkinson disease: a case-control study.

OBJECTIVE: It has been suggested that anemia may be a risk factor for dementia, for restless legs syndrome, and for Parkinson disease (PD). Thus, we investigated the association of anemia with the subsequent risk of PD using a case-control study design. METHODS: We used the medical records-linkage system of the Rochester Epidemiology Project to identify 196 subjects who developed PD in Olmsted County, Minnesota, from 1976 through 1995. Each incident case was matched by age (+/-1 year) and sex to a general population control. We reviewed the complete medical records of cases and controls in the system to detect anemia defined using the World Health Organization criteria. RESULTS: Anemia was more common in the history of cases than of controls (odds ratio 2.00, 95% confidence interval 1.31-3.06, p = 0.001). The association remained significant after adjustment for cigarette smoking, exposure to pesticides, or hysterectomy (in women). The association was not significantly different between men and women, or between PD patients with or without rest tremor. Analyses stratified by time of onset of anemia showed a greater association for anemia that started 20 to 29 years before the onset of PD. Hemoglobin levels were slightly but consistently lower in cases than in controls across all ages. CONCLUSIONS: Our results support an association between anemia experienced early in life and the later development of Parkinson disease. The interpretation of this association remains uncertain.

Potentiation of interferon-mediated inhibition of Chlamydia infection by interleukin-1 in human macrophage cultures.

One mechanism by which interferons (IFNs) can inhibit chlamydial infection is by the induction of the enzyme indoleamine 2,3-dioxygenase (IDO), which restricts the availability of tryptophan, which is required for chlamydial growth. Other immunomodulating agents, including interleukin-1 (IL-1), can interact synergistically with IFNs, resulting in increased IDO activity in macrophages. The objectives of this study were to establish that IL-1 can enhance IFN-mediated inhibition of chlamydial growth by increasing the amount of IDO activity induced by IFNs and to identify immunomodulatory agents in culture supernatants from chlamydia-infected macrophages that interact synergistically with IFNs in restricting chlamydial growth. Monocyte-derived macrophages were treated with IL-1 combined with gamma IFN (IFN-gamma) or IFN-beta. The ability of treated cells to support the growth of Chlamydia psittaci was directly related to the amount of IDO activity induced; as IDO activity increased, so did inhibition of chlamydial growth. Furthermore, concentrations of IFNs were identified at which little IDO activity was induced and chlamydial growth was permitted yet which in the presence of IL-1 resulted in increased IDO activity and restriction of chlamydial growth. The addition of exogenous tryptophan reversed the effect of combined IFN and IL-1 treatment, indicating that IDO activity induced by combined cytokine treatment was responsible for chlamydial inhibition. Supernatants from chlamydia-infected macrophages were capable of potentiating IDO induction by IFN-gamma and of restricting the growth of C. psittaci. Antibody to IL-1 beta neutralized the potentiating effects of supernatants from chlamydia-infected cells on both IDO induction and chlamydial inhibition. Thus, IL-1 produced in response to chlamydial infection may contribute to the elimination of the infection.

Transcriptional activation of indoleamine dioxygenase by interleukin 1 and tumor necrosis factor alpha in interferon-treated epithelial cells.

Interferon (IFN)-gamma-induced indoleamine 2,3-dioxygenase (IDO) activity is enhanced synergistically by interleukin (IL-)1, tumor necrosis factor-alpha (TNF-alpha) and LPS in IFN-treated macrophages by increasing IDO mRNA concentration. These studies demonstrate that IFN-treated HeLa cells also exhibit dose-dependent enhancement of IDO induction by TNF-alpha and IL-1, with maximal effects at concentrations of 5 ng/ml and 3 ng/ml, respectively. Furthermore, with sub-optimal IFN concentrations, cells treated with maximally effective concentrations of TNF-alpha or IL-1alpha required 3-5 times less IFN to induce the same level of IDO activity as that observed with IFN alone. To detect changes in transcriptional activation of the IDO gene, HeLa cells were transfected with a plasmid containing the IDO 5' regulatory region upstream of a green fluorescent protein (GFP) reporter gene. In transfected cells, IFN induced both IDO activity and GFP that was detected by flow cytometry. When cell-sorted, transfected cells were stimulated with IFN in combination with TNF-alpha or IL-1 but not LPS, increased GFP was detected in comparison to transfected cells treated with IFN alone. Furthermore, increases in GFP expression correlated with IDO enzymatic activity, indicating that combinations of IFN with IL-1 or TNF-alpha increase the transcriptional activity of the IDO promoter region.

Interferon-induced indoleamine 2,3-dioxygenase activity inhibits Chlamydia psittaci replication in human macrophages.

Interferon-gamma (IFN-gamma) previously has been shown to inhibit the replication of Chlamydia psittaci in epithelial cells by inducing indoleamine 2,3-dioxygenase, the enzyme that decyclizes tryptophan to N-formylkynurenine. The role of indoleamine 2,3-dioxygenase in IFN-mediated inhibition of C. psittaci in human macrophages has now been examined. Peripheral blood monocytes from normal donors were isolated and cultivated 10-14 days to allow differentiation to macrophages. Cells were then treated with either IFN-gamma or IFN-beta for 48 h before infection with sufficient C. psittaci to infect approximately 30% of the cells. Infected cells were incubated 24 h, at which time coverslips were fixed, stained with Giemsa, and examined for development of C. psittaci inclusions by light microscopy. Complete inhibition of inclusion development was observed with IFN-gamma. In the absence of lipopolysaccharide, inhibition of C. psittaci by IFN-beta was variable; however, in the presence of lipopolysaccharide, IFN-beta also completely inhibited C. psittaci replication. The addition of excess tryptophan to the culture medium at the time of infection partially reversed the effect of IFN on the inhibition of C. psittaci growth in a concentration-dependent manner. Indoleamine 2,3-dioxygenase activity was determined by measurement of the concentrations of tryptophan and its metabolites in the culture medium after reversed-phase high-performance liquid chromatography. Significant indoleamine 2,3-dioxygenase activity was observed only in macrophages treated with IFN-gamma or combined IFN-beta plus lipopolysaccharide, and resulted in greater than 50% of available tryptophan being catabolized in a 4-h period.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhancement of indoleamine 2,3-dioxygenase activity in cancer patients receiving interferon-beta Ser.

Degradation of tryptophan to kynurenine, catalyzed by indoleamine 2,3-dioxygenase (IDO), has been shown previously to be augmented in human peripheral blood mononuclear cells (PBMCs) treated in vitro with interferons-alpha, -beta, and -gamma (IFNs), and in human epithelial cells treated with IFN-gamma. To determine whether administration of IFN in vivo also induced IDO activity, tryptophan degradation by PBMCs purified from patients was measured. PBMCs, obtained prior to and 24 h after i.v. bolus injection of 90 X 10(6) units or 180 X 10(6) units of IFN-beta Ser, were cultivated in medium containing [3H]tryptophan. After 48 h incubation, culture supernatants were harvested, and the amount of tryptophan degraded was measured by fractionation with high-performance liquid chromatography and quantification of radioactivity in resultant fractions. Significantly enhanced IDO activity was observed after IFN-beta Ser treatment, with a mean paired increase of 43% of maximum inducible activity. Thus, measurement of in vivo induction of IDO activity may be a useful indicator in optimizing therapeutic use of IFNs in neoplastic, infectious, and other clinical disorders.

Upregulation of interferon-induced indoleamine 2,3-dioxygenase in human macrophage cultures by lipopolysaccharide, muramyl tripeptide, and interleukin-1.

The tryptophan decyclizing enzyme indoleamine 2,3-dioxygenase (IDO) was induced in human monocyte-derived macrophages (MDM) treated with human recombinant interferon-beta (IFN-beta) or interferon-gamma (IFN-gamma). Treated cells exhibited dose-dependent increases in IDO when assayed 48 hr after treatment. Cells exposed to IFN-gamma were observed to exhibit consistently higher peak levels of IDO when compared with cells incubated in the presence of IFN-beta. When IFN-beta-treated cells were incubated in the presence of specified amounts of bacterial lipopolysaccharide (LPS) or liposome-encapsulated muramyl tripeptide (MTP), peak IDO activity increased such that enzyme activity was comparable to maximal activity observed with IFN-gamma-treated cells. LPS and MTP also upregulated IFN-gamma-mediated IDO activity when suboptimal amounts of IFN-gamma were used. When macrophages were costimulated with various concentrations of human recombinant interleukin 1 alpha (IL-1 alpha), along with either maximum-stimulating amounts of IFN-beta or suboptimal amounts of IFN-gamma, IDO activity was upregulated in a manner similar to results obtained using the microbial products as stimuli. While neither IL-1 alpha or IL-1 beta was detected in culture supernatants from macrophages treated with either LPS or MTP (alone or in combination with IFN), IL-1 alpha was detected in cell lysates of macrophages treated with these upregulators. Although neutralizing antibody to IL-1 alpha abolished the upregulatory effect of exogenous IL-1 alpha, it had no effect on upregulation by LPS or MTP. This suggests that although LPS and MTP may induce production of cell-associated IL-1 alpha, upregulation of IDO activity by these agents is independent of IL-1 alpha production and may be mediated through distinct pathways.

Cytokine-mediated indoleamine 2,3-dioxygenase induction in response to Chlamydia infection in human macrophage cultures.

The purpose of this study was to characterize further the events leading to the metabolic degradation of tryptophan in Chlamydia-infected cultures in the absence of added interferon (IFN). Macrophages on coverslips were infected with Chlamydia psittaci, and tryptophan decyclization was determined 24 h later by reverse-phase high-performance liquid chromatography. Tryptophan metabolites cochromatographed with kynurenine and N-formylkynurenine, the end products of tryptophan decyclization by the IFN-inducible enzyme indoleamine 2,3-dioxygenase (IDO). Although chloramphenicol pretreatment completely inhibited chlamydial replication, IDO was stimulated to an extent similar to that in untreated, infected cells. No IDO induction was observed in cells pretreated with cycloheximide even though chlamydial growth was slightly greater than in untreated cells. These results indicate that enhanced tryptophan decyclization was due to induction of IDO. IDO induction was dependent on the size of the chlamydial inoculum. Heat- or UV-inactivated chlamydiae induced significantly less IDO activity than viable chlamydiae. Culture supernatants from Chlamydia-infected macrophages induced IDO activity in a dose-dependent manner, suggesting that a secreted product of infected cells was responsible for IDO induction. A combination of neutralizing antibodies to IFN-alpha and IFN-beta inhibited induction of IDO activity by infected cell culture supernatants. Furthermore, IL-1 enzyme-linked immunosorbent assay results indicated the accumulation of IL-1 beta in the culture medium. Thus, induction of IDO in Chlamydia-infected macrophages reflects the production of cytokines in response to infection and may represent a normal host cell response to control intracellular infection.

Beta interferon inhibits Toxoplasma gondii growth in human monocyte-derived macrophages.

Gamma interferon (IFN-gamma) previously has been shown to block the replication of Toxoplasma gondii in fibroblasts by the induction of indoleamine 2,3-dioxygenase (IDO) activity. IFN-beta also is known to induce IDO activity in monocyte-derived macrophages, but its ability to block the growth of T. gondii has not been demonstrated. We found not only that the combination of IFN-beta and lipopolysaccharide induced greater IDO activity in monocyte-derived macrophages than did IFN-beta alone but that this combination also was effective in inhibiting the growth of T. gondii. In addition, the inhibition was reversed by the addition of exogenous tryptophan, thus demonstrating that a mechanism by which IFN-beta inhibited T. gondii replication was by the induction of IDO.

Long-term effects of gamma interferon on chlamydia-infected host cells: microbicidal activity follows microbistasis.

When human monocyte-derived macrophages or a human uroepithelial cell line (T24 cells) was incubated in the presence of gamma interferon (IFN-gamma) for 24 to 48 h and then infected with Chlamydia psittaci, host cells became activated to restrict intracellular C. psittaci growth. A reversal of this inhibition was observed when infected cells were supplemented with excess exogenous tryptophan at the time of infection or at 1 to 3 days after infection. When IFN-gamma-treated, infected cells were incubated for more extended periods of time before the addition of exogenous tryptophan, no recovery of viable chlamydiae was observed. Neither replacement of the IFN-gamma-containing medium with complete medium supplemented with excess tryptophan nor the addition of excess concentrations of all 20 amino acids with and without essential vitamins contributed to the reversal of IFN-gamma-mediated inhibition of chlamydial growth beyond the time when reversal occurred after the addition of exogenous tryptophan alone. These data provide evidence which indicates that although IFN-gamma treatment of host cells initially results in a microbistatic inhibition of intracellular chlamydial development, longer incubations result in microbicidal activity that is irreversible by modulation of essential nutrient levels.

Interferons and indoleamine 2,3-dioxygenase: role in antimicrobial and antitumor effects.

Indoleamine 2,3-dioxygenase (IDO) is an interferon (IFN)-induced protein that initiates the metabolism of tryptophan along the kynurenine pathway. Although IDO can be induced by IFN-gamma in many cell types, only mononuclear phagocytes have been shown to be induced to decyclize tryptophan by all three IFN classes. Since tryptophan is an essential amino acid necessary for a variety of metabolic processes, depletion of available tryptophan may be an important mechanism for control of rapidly-dividing microbial pathogens and tumors. The purpose of this review is to present evidence that documents the effects of IFN-induced IDO on prokaryotic and eukaryotic pathogens, as well as on a variety of tumor cell lines.