The Expression Of Indoleamine2,3-dioxygenase And Its Regula-tions In Leukemia Cells

Objective:Indoleamine2,3-dioxygenase(IDO) is an extrahepatic enzyme that catalyzes the initial and ratelimiting step in the degradation of tryptophan along the kynurenine pathway. The active expression of IDO lead to deprivation of tryptophan in microenvironment, thereby kept them at a state of "Tryptophan Starvation", and cells including T cells was particularly sensitive to the state in rapidly dividing stage or pathogenic microorganisms. So IDO can inhibit the proliferation of T cells. In addition, products of tryptophan catabolism induced by IDO, such as L-kynurenine, prcolinic acid and quinolinic acid, have toxic effects on Tcells. So the upregulation of IDO expression can bring intense immunosuppressive. IDO is secreted by peripheral monocyte,macrophage and trophoblasts, which is widely expressed in human and other mammal’s extrahepatic tissues, and has high activity in tissues of lung, small intestine and placenta.Moreover, many organizations found that primary tumor cells and tumor cell lines have high expression of IDO, such as ovarian carcinoma,endometrial carcinoma,colon carcinoma,and human adult T-cell leukemia/lymphoma,which induced immune tolerance of tumor. Leukemia is malignant tumor of hematology field. New study demonstrated that AML cells,but not CD34+ hematopoietic stem/progenitor cells,express an active IDO protein,and high IDO mRNA level in blasts of acute myeloid leukemia patients predicts poor clinical outcome. The expression of IDO and its mechanisms is particularly complex as a new immunosuppressant. IDO can be induced in vitro or in vivo by various agents like cytokines(IFN-γ,TNF-α), CpG motif, CD40L,CTLA4-Ig,Influenza virus,or bacterial LPS. Moreover,thymosin alpha1(Tα1) can upregulate expression of IDO via TLR9-dependent signaling pathway. The pathway of inducing IDO can be divided into interferon-dependent signaling pathway and interferon-independent pathway. JAK-STAT pathway play an important in the former pathway, and the latter pathway has a correlation with p38MAPK pathway and NF-κB pathway. The purpose of this experiment is to study IDO expression and its regulation in leukemia cells, thereby supply theoretic evidence on the induction of immune tolerance in development or therapy process of leukemia,and provide new way to improve the immunotherapy of leukemia patients. So as a independent prediction index of leukemia patient’s clinical outcome,IDO can be used to evaluate prognosis and chemotherapy sensitivity about leukemia patient.Methods:1.Expressions of IDO and TLR9 of HL-60 cells and K562 cells cocultured with or without IFN-γ,Tα1,IFN-γ/Tα1 and chloroquine were determined by reverse transcription-polymerase chain reaction(RT-PCR).2.The total IDO protein of supernatant in HL-60 cells and K562 cells cocultured with or without IFN-γ,Tal and IFN-γ/Tα1 was assayed by the Bradford me-thod.Then IDO activity was assayed by the colorimetric method with minor modifications. Results:1.HL-60 cells and K562 cells all express IDOmRNAs and TLR9mRNAs.IFN-y and Tal respectively upregulate and downregulate the expression of IDO with concentration-dependent ways. And there are a lack of IDO mRNA in HL-60 cells as the concentration of Tal is 1μg/ml.moreover,Tal can wake the function of IFN-γon inducing IDO expression.Chloroquine has not effect on the expression of IDO.The expression of TLR9mRNA has not obviously changing in HL-60 cells and K562 cells cocultured with every group.2.There are not obviously changing of total protein in supernatant of HL-60 cells and K562 cells cocultured with IFN-γ,Tal and IFN-γ/Tα1. IFN-γcan induce high levels of IDO activity with concentration-indepent in HL-60 cells and K562 cells.On the contrary Tal can incoordinately inhibit IDO activity of HL-60 cells and K562 cells. And Tα1 can obviously waken the role of IFN-γon IDO activity. Conclusion:1.IDO express on leukemia cells and has activity, which indicate that IDO can degradate tryptophan of leukemia cells and then inhibit T cell proliferation and activation.As a new immunosuppressor,IDO maybe play an important in immune tolerance induced by leukemia cells.Then as a independent prediction index of leukemia patient’s clinical outcome,IDO can be used to evaluate prognosis and sensitivity of chemotherapy about leukemia patients.2.IFN-γcan increase IDO expression and activity of leukemia cells,suggesting that IFN-γmaybe also make tumor cells escape attack from immune system as a result of proliferation of tumor cells,when it play a therapeutic role in leukemia or other tumor.But Tα1 can decrease IDO expression and activity of leukemia cells and waken the role of IFN-γon expression and activity of IDO,which implies that as a immunoregulatory,Tα1 can break the state of immune tolerance inducing by leukemia cells and then improve effect of immune/chemical treatment and prognosis of leukemia as a adjunct to chemotherapy.3.Leukemia cells express IDO and TLR9.IFN-γand Tα1 respectivly induce and reduce the expression of IDO.But the expression of TLR9 has no significant change on leukemia cells cocultured with IFN-γand Tα1, suggesting that the regulation of IFN-γand Tα1 on the expression of IDO maybe has no relationship with TLR9 signaling pathways.