Raymond R. Brown

Implications of Interferon-Induced Tryptophan Catabolism in Cancer, Autoimmune Diseases and Aids

Tryptophan (Trp) is an indispensable amino acid required for biosynthesis of proteins, serotonin and niacin. Indoleamine 2,3-dioxygenase (IDO) is induced by infections, viruses, lipopolysaccharides, or interferons (IFNs) and this results in significant catabolism of Trp along the kynurenine (Kyn) pathway. Intracellular growth of Toxoplasma gondii and Chlamydia psittaci in human fibroblasts in vitro is inhibited by IFN-gamma and this inhibition is negated by extra Trp in the medium. Similarly, growth of a number of human cell lines in vitro is inhibited by IFN-gamma and addition of extra Trp restores growth. Thus, in some in vitro systems, antiproliferative effects of IFN-gamma are mediated by induced depletion of Trp. We find that cancer patients given Type I or Type II IFNs can induce IDO which results in decreased serum Trp levels (20-50% of pretreatment) and increased urinary metabolites of the Kyn pathway (5 to 500 fold of pretreatment). We speculate that in vivo antineoplastic effects of IFNs and clinical side effects are mediated, at least in part, by a general or localized depletion of Trp. In view of reported increases of IFNs in autoimmune diseases and our earlier findings of elevated urinary Trp metabolites in autoimmune diseases, it seems likely that systemic or local depletion of Trp occurs in autoimmune diseases and may relate to degeneration, wasting and other symptoms in such diseases. We find high levels of IDO in cells isolated from synovia of arthritic joints. IFNs are also elevated in human immunodeficiency virus (HIV) patients and increasing IFN levels are associated with a worsening prognosis. We propose that IDO is induced chronically by HIV infection, is further increased by opportunistic infections, and that this chronic loss of Trp initiates mechanisms responsible for the cachexia, dementia, diarrhea and possibly immunosuppression of AIDS patients. In these symptoms, AIDS resembles classical pellagra due to dietary deficiency of Trp and niacin. In preliminary studies, others report low levels of Trp and serotonin, and elevated levels of Kyn and quinolinic acid in AIDS patients. The implications of these data in cancer, autoimmune diseases and AIDS are discussed.

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-γ 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.

Determination of tamoxifen and metabolites in human serum by high-performance liquid chromatography with post-column fluorescence activation

Sensitive and reproducible analyses were developed for assaying tamoxifen, monohydroxytamoxifen, N-desmethyltamoxifen, metabolite E [trans-1(4-hydroxyphenyl)1,2-diphenylbut-1-ene] and a new metabolite, metabolite Y [trans-1(4-hydroxyethoxyphenyl)-1,2-diphenylbut-1-ene] in human serum using high-performance liquid chromatography (HPLC). Three different systems were developed for specific purposes. All chromatography was performed using serum extracts made with hexane-butanol. Detection was by fluorimetry of phenanthrene derivatives formed by on-stream UV irradiation with a newly described device for post-column irradiation of the HPLC stream. This device may be of use in other HPLC systems requiring post-column photochemical reactions.

Relationship between tryptophan metabolism and heterotopic recurrences of human urinary bladder tumors

In order to investigate the possible relationship between tryptophan metabolism and heterotopic recurrences of human bladder tumors, tryptophan metabolite excretion patterns of 38 patients with low‐stage bladder tumors were analyzed before and after the ingestion of L‐tryptophan. Periodic cystoscopic examinations were performed after the initial removal of bladder tumors. Among 38 patients, 20 patients had normal tryptophan metabolism, and 18 patients had abnormal tryptophan metabolism. Follow‐up studies disclosed that all of 18 patients with abnormal tryptophan metabolism had one or more recurrences within 5 years; however, among 20 patients with normal tryptophan metabolism, 8 patients had no recurrences for 5 years, and 12 patients had recurrences within 5 years. These data suggest that tryptophan metabolites may play a role in heterotopic recurrences of bladder tumors in patients with abnormal tryptophan metabolism and that it might be possible to retard the development of recurrences by correction of tryptophan metabolism in those patients.

Inhibition of collagenase and stromelysin gene expression by interferon-gamma in human dermal fibroblasts is mediated in part via induction of tryptophan degradation.

The expression of the matrix-degrading enzymes collagenase and stromelysin is modulated by a variety of biologic and pharmacologic agents. IFN-gamma has potent effects on metalloproteinase production and therefore may play an important role in preventing excessive connective tissue degradation during inflammation and repair. We investigated the mechanisms of collagenase and stromelysin regulation by IFN-gamma in human dermal fibroblasts. IFN-gamma (300 U/ml) prevented the stimulation of metalloproteinase gene expression by IL-1 beta. In addition, incubation of fibroblasts with IFN-gamma resulted in a marked increase in cellular indoleamine 2,3-dioxygenase (IDO) mRNA, a > 90% depletion of tryptophan, and a corresponding > 30-fold increase in the tryptophan metabolite kynurenine in the culture media. Reducing the concentration of tryptophan from 25 microM to 0 markedly diminished the ability of fibroblasts to increase collagenase and stromelysin mRNA and collagenase production in response to IL-1 beta. Addition of exogenous tryptophan (25-50 micrograms/ml) to cultures that had been tryptophan depleted by pretreatment with IFN-gamma for 48 h restored the fibroblast response to IL-1 beta or PMA, but had no effect on IFN-gamma-induced HLA-DR alpha chain mRNA expression. These results indicate that inhibition of collagenase and stromelysin gene expression by IFN-gamma in fibroblasts is associated with activation of IDO and enhanced cellular tryptophan metabolism. Tryptophan degradation and ensuing tryptophan depletion may account, at least in part, for the inhibitory effect of IFN-gamma on metalloproteinase production in dermal fibroblasts.

A direct comparison of biological response modulation and clinical side effects by interferon-beta ser, interferon-gamma, or the combination of interferons beta ser and gamma in humans.

To directly compare clinical side effects and biological response modification, IFN-beta ser, IFN-gamma, or the combination of IFN-beta ser plus IFN-gamma was administered to 21 cancer patients. Each IFN or the combination was given intravenously on days 1, 8, and 15 in varied order. Each IFN and the combination resulted in significant (P less than 0.05) modulation of IFN-induced proteins. IFN-beta ser was more effective than IFN-gamma in enhancing 2-5A synthetase activity (P = 0.001). IFN-gamma was more effective than IFN-beta ser in enhancing serum beta 2 microglobulin expression (P = 0.05) and indoleamine dioxygenase activity, as assessed by decreased serum tryptophan (P = 0.03). The combination enhanced tryptophan catabolism more effectively than IFN-beta ser in a dose-dependent manner (P less than 0.03). IFN-beta ser/IFN-gamma did not potentiate natural killer cells or antibody-dependent cellular toxicity (ADCC). IFN-beta ser/IFN-gamma enhanced monocyte guanylate cyclase activity, as assessed by serum neopterin, more effectively than IFN-gamma alone (P = 0.005). Both IFNs and the combination resulted in increases in HLA class II expression on monocytes. However, no significant difference in the level of induction of HLA DQ and HLA DR expression between IFN-beta ser/IFN-gamma and either IFN-beta ser or IFN-gamma was noted. Although frequency and servity of side effects of IFN-beta ser, IFN-gamma, or the combination were dose related, induction of induced proteins (with exception of influences on tryptophan catabolism) were not a function of dose administered over the 10-fold range. Continued treatment with the combination intravenously three times a week for 4 wk sustained but did not further potentiate, most of the changes in interferon-induced proteins. Thus, IFN-beta ser and IFN-gamma each resulted in effective and essentially equivalent patterns of induction of induced proteins. When combined, however, these IFNs did not result in potentiation of biological response modification in vivo.

Tryptophan metabolism in breast cancer. correlation with urinary steroid excretion

Urinary excretion of tryptophan metabolites, etiocholanolone, androsterone, and 17‐hydroxycorticosteroids (17‐OHc.s.), was measured in 25 patients with breast cancer and in 12 similarly aged control women. Twelve of the 25 patients had elevated excretions of metabolites of the kynurenine‐niacin pathway, and, in these patients, the excretion of etiocholanolone was significantly lower than in controls or in patients with normal tryptophan metabolism.

Clinical and biologic effects of combination therapy with gamma‐interferon and tumor necrosis factor

Tumor necrosis factor (TNF) and gamma‐interferon (γ‐IFN) are cytokines with synergistic biologic and antiproliferative effects in vitro and in mouse models. The biologic effects of the combination of TNF and γ‐IFN, however, have not been studied well in humans. A Phase I trial was conducted of TNF and γ‐IFN therapy in 24 patients with advanced malignancies to determine the tolerability of the combination and the biologic effects of TNF and γ‐IFN in vivo. Both TNF and γ‐IFN were administered as 30‐minute intravenous infusions three times per week. Doses of TNF ranged from 25 to 100 μg/m2; all patients received 100 μg/m2 of γ‐IFN. Dose‐limiting toxicity consisted primarily of orthostatic hypotension and constitutional symptoms. The maximum tolerated dose level (MTDL) of 50 μg/m2 of TNF and 100 μg/m2 of IFN‐γ was less than the maximum tolerated dose (MTD) observed in previous Phase I trials of γ‐IFN and TNF alone. Biologic responses were studied in seven patients treated at the MTDL. Serum interleukin‐2 receptor levels and neopterin secretion were enhanced significantly 24 hours after therapy (P = 0.002); enhancement of monocyte Fc receptor levels had borderline statistical significance (P = 0.07). With the exception of the mean fluorescent intensity on monocytes positive for histocompatibility antigen HLA‐DR (P = 0.03), HLA Class I and II cell surface protein expression was not increased. The combination significantly enhanced indoleamine dioxygenase activity and serum β2‐microglobulin expression (P < 0.04) but not 2′,5′‐oligoadenylate synthetase activity, bactericidal function, or chemiluminescence. These results were compared retrospectively with those observed in previous Phase I trials of γ‐IFN and TNF alone. The combination of TNF and γ‐IFN significantly increased urinary kynurenine levels more than either TNF alone or γ‐IFN alone. Given the limitations inherent in any retrospective analysis, however, the enhancement in the other biologic parameters measured at the MTDL during this trial did not differ significantly from the changes observed at the MTD of either TNF or γ‐IFN alone. It was concluded that the combination of TNF and γ‐IFN, when administered at the MTDL of the combination, does not offer any enhancement in biologic responses over either agent alone.

Direct and steam distillation autoanalyzer methods for assay of diazotizable aromatic amine metabolites of tryptophan in urine and in serum.

Abstract A procedure is described for the assay of aromatic amine metabolites of tryptophan by an automated diazotization and coupling procedure using Technicon Autoanalyzer equipment. The assay is reliable and sensitive and if many samples are to be analyzed, will afford a several-fold saving in time and labor. This automated procedure is also satisfactory for assay of the enzymes tryptophan oxygenase and kynureninase. In addition, a procedure is described for automated steam distillation of aromatic amines in urine and serum and for their simultaneous assay by means of diazotization and coupling. This steam distillation method is specific for kynurenine and acetylkynurenine and is suitable for the routine screening of large numbers of urine or blood serum samples for elevated levels of these metabolites as a measure of abnormal tryptophan metabolism.

Tryptophan metabolism and corticosteroids in breast cancer

Urinary excretion of tryptophan metabolites, free cortisol, and corticosteroid sulfates, and plasma cortisol were determined in 30 breast cancer patients. Although, as a group, the patients with abnormal tryptophan metabolism excreted significantly higher levels of both free cortisol and corticosteroid sulfates than those with normal metabolism, there was no good correlation between urinary tryptophan metabolite excretion and the corticoids. Abnormal tryptophan metabolism was not accompanied by a high plasma cortisol. Patients with widespread metastases had elevated tryptophan metabolite excretions compared to those with early breast cancer or local recurrence, but the two groups did not differ in their plasma or urinary corticosteroid levels. It is concluded that increased adrenocorticosteroid secretion due to stress is not a major factor in producing abnormal tryptophan metabolism in breast cancer.