Masao Takemura

Plasma Fas Ligand, an Inducer of Apoptosis, and Plasma Soluble Fas, an Inhibitor of Apoptosis, in Patients With Chronic Congestive Heart Failure

OBJECTIVES This study sought to examine plasma levels of soluble Fas/APO-1 receptor (sFas), an inhibitor of apoptosis, and soluble Fas ligand (sFas-L), an inducer of apoptosis, and their relation to each other and to other clinical variables, such as New York Heart Association functional class, tumor necrosis factor (TNF) and interleukin-6 (IL-6) in congestive heart failure (CHF). BACKGROUND It has been recently reported that apoptotic cell death occurs in myocytes of dogs with CHF. Hypoxia is frequently seen in advanced CHF and can stimulate Fas/APO-1 receptors (Fas) to induce apoptosis in cultured myocytes. Fas and Fas ligand (Fas-L) are cell-surface proteins and representative apoptosis-signaling molecules. Fas on the cell membrane induces apoptosis when it binds Fas-L or sFas-L. However, plasma sFas, a molecule lacking the transmembrane domain of Fas, blocks apoptosis by inhibiting binding between Fas and Fas-L or sFas-L on the cell membrane. At present, it is unknown whether plasma sFas-L and plasma sFas increase in the presence of cardiac disease. METHODS The study included 70 patients (mean [+/-SEM] age 65 +/- 2 years, range 21 to 93) with chronic CHF (coronary artery disease in 28, dilated cardiomyopathy in 27, valvular heart disease in 15) and 62 age- and gender-matched normal control subjects. Plasma levels of sFas, sFas-L, TNF-alpha and IL-6 were measured by enzyme-linked immunosorbent assays using monoclonal anti-human antibodies. RESULTS There was no significant difference in sFas-L levels between normal subjects and patients in functional classes I to IV; however, sFas increased with severity of functional classification, independent of the underlying disease. sFas levels were significantly higher even in patients in functional class II than in normal subjects and those in functional class I, and were highest in patients in functional class IV (normal subjects; 2.2 +/- 0.1 ng/ml; functional class I: 2.2 +/- 0.2 ng/ml; functional class II: 3.1 +/- 0.2 ng/ml; functional class III: 3.9 +/- 0.3 ng/ml; functional class IV: 5.1 +/- 0.6 ng/ml). Plasma sFas levels were significantly higher in patients with elevated pulmonary artery wedge pressure and a decresed cardiac index than in those with values in the normal range. In patients in functional class IV, there was no significant difference in plasma sFas levels between the survivors and non-survivors during 6-month follow-up. However, plasma levels of sFas tended to decrease in nine patients with clinical improvement (baseline sFas: 5.2 +/- 0.8 ng/ml; 6-month sFas: 4.3 +/- 0.5 ng/ml, p = 0.07) but were similar in patients with no change in functional class. TNF-alpha and IL-6 were increased significantly only in patients in functional class IV, as previously reported, but were not related to sFas. CONCLUSIONS We found elevated levels of plasma sFas and no increase in plasma sFas-L in human CHF. The increase in sFas may play an important role in the pathophysiologic mechanisms of CHF.

L-tryptophan-L-kynurenine pathway metabolism accelerated by Toxoplasma gondii infection is abolished in gamma interferon-gene-deficient mice: cross-regulation between inducible nitric oxide synthase and indoleamine-2,3-dioxygenase.

ABSTRACT l-Tryptophan degradation by indoleamine 2,3-dioxygenase (IDO) might have an important role in gamma interferon (IFN-γ)-induced antimicrobial effects. In the present study, the effects of Toxoplasmagondii infection on IDO were investigated by using wild-type and IFN-γ-gene-deficient (knockout) (IFN-γ KO) mice. In wild-type C57BL/6J mice, enzyme activities and mRNA levels for IDO in both lungs and brain were markedly increased and lung l-tryptophan concentrations were dramatically decreased following T. gondii infection. In contrast, these metabolic changes did not occur in T. gondii-infected IFN-γ KO mice or in uninfected IFN-γ KO mice. The levels of inducible nitric oxide synthase (iNOS) induction in infected IFN-γ KO mice were high in lungs and low in brain compared to those in infected wild-type mice. The extent of increased mRNA expression of T. gondii surface antigen gene 2 (SAG2) induced in lungs and brain by T. gondii infection was significantly enhanced in IFN-γ KO mice compared to wild-type mice on day 7 postinfection. Treatment with N-nitro-l-arginine methyl ester, an iNOS inhibitor, increased the levels of SAG2 mRNA in brain but not in lungs and of plasma l-kynurenine after T. gondii infection. This in vivo study provides evidence that l-tryptophan depletion caused by T. gondii is directly mediated by IFN-γ in the lungs, where iNOS is not induced by IFN-γ. This study suggests that there is an antitoxoplasma mechanism of cross-regulation between iNOS and IDO and that the expression of the main antiparasite effector mechanisms for iNOS and/or IDO may vary among tissues.

3-Hydroxyanthranilic acid, an L-tryptophan metabolite, induces apoptosis in monocyte-derived cells stimulated by interferon-γ

3-Hydroxyanthranilic acid (3-HAA), a metabolite of L-tryptophan, accumulates in monocyte-derived cells (THP-1),but not in other celllines tested(MRC9, H4, U373MG, Wil-NS), following immune stimulation that induces indoleamine 2,3-dioxygenase (IDO), a rate-limiting enzyme in the L-tryptophan-kynurenine pathway. We examined whether metabolites of the L-tryptophan-kynurenine pathway act to induce apoptosis in monocytes/macrophages. Of the L-tryptophan metabolites tested, only 3-HAA at a concentration of 200µmol/L was found to induce apoptosis in THP-1 and U937 cells. The addition of ferrous or manganese ions further enhanced apoptosis and free radical formation by 3-HAA in these two types of cells. The apoptotic response induced by 3-HAA was significantly attenuated by the addition of antioxidant, α-tocopherol or Trolox (a water-soluble analogue of vitamin E), and the xanthine oxidase inhibitor, allopurinol. In addition, the 3-HAA-induced apoptotic response was slightly attenuated by catalase, but not by superoxide dismutase (SOD), indicating that generation of hydrogen peroxide is involved in this response. Interferon-γ (IFN-γ), an inducer of IDO, potently induced apoptosis in THP-1 cells, but not in U937 cells, in the presence of ferrous or manganese ions. This different susceptibility to apoptosis inducer between THP-1 and U937 cells may depend on the capacity of the cells for 3-HAA synthesis following IDO induction by IFN-γ. Furthermore, apoptosis was suppressed by cycloheximide in THP-1 cells, suggesting that newly synthesized proteins may be essential for apoptotic events. These results suggest that 3-HAA induces apoptosis in monocytes/macrophages under inflammatory or other pathophysiological conditions.

The Absence of IDO Upregulates Type I IFN Production, Resulting in Suppression of Viral Replication in the Retrovirus-Infected Mouse

Indoleamine 2,3-dioxygenase, the l-tryptophan–degrading enzyme, plays a key role in the powerful immunomodulatory effects on several different types of cells. Because modulation of IDO activities after viral infection may have great impact on disease progression, we investigated the role of IDO following infection with LP-BM5 murine leukemia virus. We found suppressed BM5 provirus copies and increased type I IFNs in the spleen from IDO knockout (IDO−/−) and 1-methyl-d-l-tryptophan–treated mice compared with those from wild-type (WT) mice. Additionally, the number of plasmacytoid dendritic cells in IDO−/− mice was higher in the former than in the WT mice. In addition, neutralization of type I IFNs in IDO−/− mice resulted in an increase in LP-BM5 viral replication. Moreover, the survival rate of IDO−/− mice or 1-methyl-d-l-tryptophan–treated mice infected with LP-BM5 alone or with both Toxoplasma gondii and LP-BM5 was clearly greater than the survival rate of WT mice. To our knowledge, the present study is the first report to observe suppressed virus replication with upregulated type I IFN in IDO−/− mice, suggesting that modulation of the IDO pathway may be an effective strategy for treatment of virus infection.

Non‐steroidal anti‐inflammatory drugs inhibit cellular proliferation and upregulate cyclooxygenase‐2 protein expression in endometrial cancer cells

We determined the effects of several non‐steroidal anti‐inflammatory drugs (NSAIDs), aspirin (acetylsalicylic acid, ASA), indomethacin and a cyclooxygenase‐2 (COX‐2)‐selective inhibitor (NS398), on cellular proliferation and regulation of COX‐2 protein expression in endometrial cancer cells in vitro, and investigated their modes of action. All three NSAIDs markedly inhibited the proliferation of Ishikawa, HEC‐1A and AN3CA endometrial cancer cell lines in a time‐ and concentration‐dependent manner. ASA and indomethacin triggered apoptosis in cells of all three lines through release of cytosolic cytochrome c, activation of caspase‐9 and ‐3, and cleavage of poly(ADP‐ribose) polymerase (PARP), but NS398 induced minimal apoptosis only in Ishikawa cells. ASA altered the cell cycle distribution, with G2/M phase accumulation of cells, and induced overexpression of Ki‐67 protein. Both ASA and indomethacin reduced the protein levels of Bcl‐2 and Bcl‐xl, but upregulated those of Bax and Bcl‐xs. COX‐2 protein expression and PGE2 production were upregulated by ASA and indomethacin in all three cell lines. However, NS398 did not alter COX‐2 protein expression or PGE2 production in these cells. These results indicate that NSAIDs inhibit proliferation of endometrial cancer cells independently of the reduction of COX‐2 protein expression. A cytochrome c‐dependent apoptotic pathway and/or cell cycle arrest may contribute to the inhibitory effects of these NSAIDs.

Acyclic retinoid induces partial differentiation, down-regulates telomerase reverse transcriptase mRNA expression and telomerase activity, and induces apoptosis in human hepatoma-derived cell lines.

BACKGROUND/AIMS Acyclic retinoid (AR; all trans-3,7,11,15-tetramethyl-2,4,6,10,14-hexadecapentaenoic acid) prevented hepatocarcinogenesis in animal models and in a randomized clinical trial by eradicating premalignant and latent malignant clones of transformed cells from the liver. We investigated the possible mechanism of this clonal deletion at the cellular level. METHODS Human hepatoma-derived cell lines, PLC/PRF/5, HuH-7, and JHH-7, were treated in vitro with AR. Secretion of albumin and that of lectin-reactive isoform of alpha-fetoprotein (AFP-L3) were measured as markers of differentiation and dedifferentiation of the cells, respectively. Telomerase reverse transcriptase (TERT) mRNA expression and telomerase activity were measured by reverse transcriptase polymerase chain reaction (RT-PCR) and stretch PCR assay, respectively. Caspase activities were measured by colorimetric protease assay. Mitochondrial membrane permeability transition was examined by Rhodamine staining. RESULTS Production of albumin was recovered while that of AFP-L3 was reduced after exposure of the cells to 10 microM AR for 2 days. This differentiation was maintained for another 2 days without retinoid. In parallel, both TERT mRNA expression and telomerase activity were down-regulated. The cells subsequently died due to apoptosis after 4-6 experimental days. Serial increases in mitochondrial membrane permeability and caspase-9 and -3 activities induced apoptosis. CONCLUSIONS AR first induces differentiation and reduces telomerase activity. Subsequent apoptosis may contribute to the eradication of the clone.

Serum concentration of L-kynurenine predicts the clinical outcome of patients with diffuse large B-cell lymphoma treated with R-CHOP

Purpose: Introduction of rituximab has largely improved the prognosis of patients with diffuse large B‐cell lymphoma(DLBCL). Such change in therapeutic outcome necessitates the identification of additional prognostic factors to conventional indexes that have been validated for CHOP without rituximab. Indoleamine 2,3‐dioxygenase (IDO) exerts intense immunomodulatory effects because of enzymatic activities that catalyze the breakdown of the essential amino acid L‐tryptophan. The activity of IDO can be estimated by measuring the serum concentration of L‐kynurenine. Here, we investigated the role of L‐kynurenine as a prognostic marker in R‐CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisolone) therapy. Experimental design: Data from 73 consecutive patients treated with eight cycles of R‐CHOP or R‐THP (tetrahydropyranyl adriamycin)‐COP between December 2002 and March 2007 were analyzed. L‐kynurenine concentrations in serum samples obtained at admission were measured by high‐performance liquid chromatography. Results: The median serum L‐kynurenine level was 1.575 μm (range 0.537–9.588). The complete response (CR) rates of patients with L‐kynurenine <1.5 and ≥1.5 μm were 83% and 61%, respectively (P < 0.05). The three‐yr overall survival (OS) rates for patients with L‐kynurenine <1.5 and ≥1.5 μm were 89% and 58%, respectively (P < 0.005). In addition, higher age, poor performance status, elevated serum lactate dehydrogenase, and unfavorable as well as revised International Prognosis Index were significantly worse factors for CR rate and OS. Multivariate analyses revealed only L‐kynurenine as an independent prognostic factor for OS. Conclusions: Serum L‐kynurenine might be a novel prognostic factor to determine the treatment outcome of DLBCL with the R‐CHOP regimen.

Indoleamine 2,3-dioxygenase is highly expressed in human adult T-cell leukemia/lymphoma and chemotherapy changes tryptophan catabolism in serum and reduced activity

Adult T-cell leukemia/lymphoma (ATLL) is caused by human T-cell lymphotropic virus type 1 (HTLV-1). Indoleamine 2,3-dioxygenase (IDO), the l-tryptophan (l-TRP)-degrading enzyme, plays a key role in the powerful immunomodulatory effects of several different types of immune cells. In this study, we investigated the IDO expression in ATLL cells and the effect of chemotherapy on IDO-initiating l-TRP catabolism in patients with ATLL. Serum l-kynurenine (l-KYN) concentrations, l-KYN/l-TRP ratio, and the level of IDO mRNA expression in ATLL cells were significantly increased in ATLL patients compared to those in healthy and HTLV-positive carrier subjects. On the other hand, l-TRP level was significantly decreased in ATLL patients compared to that in healthy subjects. In the immunohistochemical staining, IDO was strongly expressed in cytoplasm of ATLL cells. Interestingly, serum l-KYN as well as soluble IL-2 receptor concentrations was significantly reduced, and l-TRP concentrations were significantly increased after chemotherapy. These data provide evidence that IDO is highly expressed in ATLL cells, and that IDO-initiating l-TRP catabolism changes with chemotherapy.

Serum-Soluble Fas Level Determines Clinical Symptoms and Outcome of Patients With Aggressive Non-Hodgkin's Lymphoma

Soluble Fas (sFas) blocks apoptosis induced by Fas ligand in vitro. The serum concentration of sFas is elevated in lympho‐proliferative diseases. We hypothesized that higher levels of sFas worsen the clinical symptoms and outcome of patients with aggressive non‐Hodgkins lymphoma (NHL). We prospectively measured the serum concentrations of sFas in 67 consecutive patients with aggressive NHL (59 with diffuse large cell lymphoma and 8 with diffuse small cleaved cell lymphoma). sFas was significantly elevated in patients with aggressive NHL compared to healthy controls (N = 36, P< 0.005), while sFas in patients with B symptoms (4.20 ± 2.12 μg/l) was significantly higher than in those without B symptoms (2.66 ± 1.08 μg/l, P < 0.005). No significant difference was observed between B‐cell lymphoma and T‐cell lymphoma or between patients with clinical stage I or II and those with clinical stage III or IV. Significant correlations were found between sFas concentration and both soluble interleukin‐2 receptor (R = 0.400, P < 0.001) and C‐reactive protein (R = 0.340, P < 0.01) levels in patients with aggressive NHL. No correlation was observed between sFas and either white blood cell count or lactate dehydrogenase. Generalized Wilcoxon analysis revealed that NHL patients with sFas less than 4 μg/l had better overall survival than those with sFas above 4 μg/l (P < 0.001). The serum concentration of sFas might be associated with clinical symptoms and the prognosis of patients with aggressive NHL. Am. J. Hematol. 64:257–261, 2000.

Reduced IFNγ production in response to IL‐12 stimulation and/or reduced IL‐12 production in atopic patients

Several studies have shown that interleukin (IL)‐4 and interferon‐gamma (IFNγ) are important for the regulation of IgE production and that IL‐12 induces IFNγ.